An in vitro bioassay for steroidogenesis-stimulating activity (SSA) in charcoal-exracted rat testicular interstitial fluid (IF) was developed. The bioassay was based upon stimulation of testosterone production by Percoll gradient-purified adult rat Leydig cells during a 20 h incubation in the presence of a maximally stimulating dose of human CG (hCG). The hCG-stimulated conditions were employed to avoid assay interference by endogenous LH in the sample preparations. The standard preparation (a pool of IF from normal adult rats) stimulated testosterone production 3- to 4-fold above that obtained with a maximal dose of either hCG or LH alone, and a linear log dose-testosterone response was observed over the range from 150% to 300% of hCG-stimulated testosterone production. The bioassay had a mean index of precision (X) of 0.13 (n = 10 assays), a between assay variation of 10.7-11.7%, and a useful working range from 4.9-28 µl IF including at least three serial half-dilutions. Parallelism with the IF standard was obtained with IF collected from aged (>15 months old) rats, adult rats made bilaterally cryptorchid for either 4 weeks or 12 months, or injected 6 h previously with 100 IU hCG, and with ovine testicular lymph. Testicular SSA was not affected by coincubation with rat LH antiserum and was not attributable to prevention of oxygen-mediated enzyme damage during the incubation period. Although charcoal-exracted rat serum log dose-response relationships were nonparallel with the standard, serum displayed an apparent relative bioactivity of approximately 20% that of normal IF based on ED50 comparisons. The activity of testicular IF was not affected by coincubation with serum. Untreated and charcoal-extracted rat albumin, at an assay concentration equivalent to that present in normal rat serum or IF, caused only a minor stimulation of testosterone production. Charcoal-extracted bovine albumin, ovalbumin, epidermal growth factor, insulin-like growth factor-1, bovine 31 kDa inhibin, and transforming growth factor-β were inactive. Activity was nondetectable in rat thoracic duct lymph or high speed supernatants of adult rat testicular extracts. Testicular SSA and serum from normal rats displayed slightly different time-courses of action, with SSA active during both acute (1.0 h) and longer term (2.0-20 h) incubations, while serum stimulated testosterone production only in the longer term incubations. Both SSA and the apparent activity in serum from normal rats were due to factors with a large mol wt (Mr > 30, 000) based on ultrafiltration. Both activities were heat labile, although residual (30.3 ± 21.1%) activity remained in IF even after heating at 100 C for 30 min. These data establish the validity and usefulness of the bioassay for quantitating SSA in rat testicular IF, and possibly other biological fluids. The characteristics and relatively high levels of testicular SSA, in comparison with serum or lymph, indicate that SSA and the apparent serum activity are due to different factors, and that SSA is most likely of testicular origin.