It has been shown that, in streptozotocin diabetic rats, protamine-retarded insulin administered in vivo stimulates intimal hyperplasia in balloon-injured carotid artery. The aim of this study was to evaluate the influence of protamine on cultured human vascular smooth muscle cells (hVSMC), by observing its effects on adhesion, chemotaxis and proliferation. hVSMC were isolated during abdominal surgery, cultured and utilized at passages 6–10. We observed that protamine stimulates: 1) cell adhesion in the concentration range 0.04–20 μg/ml (analysis of variance, ANOVA, p < 0.0001); 2) cell chemotaxis in the absence of fetal calf serum (FCS) in the concentration range 1–200 μg/ml (ANOVA, p < 0.0001) and in the presence of 1 % FCS in the concentration range 5–200 μg/ml (ANOVA, p < 0.0001), further enhancing the chemotaxis induced by 10 % FCS in the concentration range 20–200 μg/ml (ANOVA, p < 0.0001); 3) cell proliferation and ³H-thymidine incorporation from 1 to 5 μg/ml (ANOVA, p < 0.0001); 4) cell c-fos oncoprotein nuclear expression. We also observed that protamine effects on chemotaxis, proliferation and c-fos expression are inhibited by heparin that human insulin stimulates cell proliferation and ³H-thymidine incorporation (ANOVA, p < 0.0001) at concentrations equal to or greater than 480 pmol/l and that these effects of insulin persist in the presence of protamine. In conclusion, protamine influences hVSMC behaviour by interfering with biological functions involved in atherogenesis. The concentrations used in this short-term in vitro study were higher than those probably occurring in vivo in patients chronically treated by protamine-retarded insulin preparations: further studies, therefore, are needed to evaluate the safety of protamine as a retardant of insulin action in vivo. [Diabetologia (1997) 40: 67–75]