The anti-proliferative effects of heparin on subcultured endothelial cells (EC) of rat aorta were investigated to determine whether heparin inhibits the competence phase or the progression phase in the cell cycle using the starting time and the rate of proliferation. Fetal bovine serum (FBS)-stimulated EC proliferation increased to 1.5-times in cell number, but decreased to 0.5-times in 3H-thymidine incorporation, compared with the proliferation of rat primary cultured smooth muscle cells (SMC). The FBS-effects on EC proliferation were attributed to the progression phase rather than the competence phase. Heparin (1, 10 or 100 micrograms/ml) significantly inhibited the proliferation of FBS (5%)-stimulated EC, and the extent of inhibition was the same in both cell number and 3H-thymidine incorporation. In the 3H-thymidine incorporation every 3 hr, heparin reduced the rate of incorporation into G0-arrested EC, but did not affect the starting time of DNA synthesis. When the index of competence phase, starting time, was plotted against that of progression phase, rate of proliferation, the inhibition of heparin was attributed to the progression phase. These results demonstrate that heparin selectively inhibits the progression phase in subcultured EC of rat aorta.