The in vitro increase in plasma levels of non-esterified fatty acids (NEFA) has been investigated in non-heparinized subject groups of differing lipoprotein lipase activity and degree of lipaemia. The influence of standard blood and plasma collection and equilibrium dialysis techniques on the extent of lipolysis has been assessed. Marked increases in plasma NEFA level occurred during equilibrium dialysis. Respective mean increases of 85% and 230% were observed in plasma collected from non-pregnant (fasted and non-fasted) and parturient subjects after dialysis at 37°C for 26 h. On the in vitro addition of ammonium heparin to blood, no difference (P > 0.05) in plasma NEFA was detected between heparin concentrations of 12.5 IU/ml and 41.7 IU/ml. In pregnant and non-pregnant subjects, no alteration (P > 0.05) in plasma NEFA levels was noted on storage of blood and plasma in ice or at room temperature for periods of up to 2h. Spurious binding estimates, however, may be a direct result of in vitro lipolysis for those drugs displaced from binding sites by NEFA and/or avidly bound to lipoproteins. Perturbation to the human serum albumin binding of diazepam (400 ng/ml) and ibuprofen (20 μg/ml) has been demonstrated at NEFA concentrations spanning those induced in vitro during equilibrium dialysis. The results of this study may also explain several anomalies and discrepancies in reported plasma binding estimates.