The presence of testicular pro-opiomelanocortin (POMC) mRNA and POMC-derived peptides has recently been demonstrated in purified preparations of interstitial macrophages and in Leydig cells of the adult rat testis by Northern blot analysis and immunocytochemistry. In the present study, in situ hybridization provided further evidence that the POMC gene is expressed by both purified interstitial macrophages and Leydig cells. The cellular localization of the POMC transcripts was similar for both cell types, silver grains being predominantly located in the cytoplasm. The specificity of the labelling was demonstrated by the lack of silver grains in the preparations pretreated with RNAase or hybridized with an insulin cDNA probe, a gene known not to be expressed in these cell types. An additional control was provided by hybridization with a sense POMC RNA probe, which gave a less intense signal when compared with the antisense RNA probe under the same experimental conditions. The results confirm POMC gene expression in both macrophages and Leydig cells in the adult rat testis.