Rapamycin, an immunosuppressant and antiproliferative agent, reduces intimal hyperplasia after arterial injury in animal models and in a preliminary study in humans. Rapamycin treatment reportedly increases expression of p27, a cyclin-dependent kinase inhibitor. This mechanism was tested using a p27-deficient (p27−/−) murine model. Aortic smooth muscle cells from wild-type (WT) and p27−/− mice were isolated and cultured. Cell proliferation, assessed by cell count and 3 H-thymidine incorporation, was inhibited significantly by rapamycin in WT and p27−/− cells at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml (p< 0.05, versus control). The in vivo effect on intimal hyperplasia was studied in p27−/− and WT mice after femoral artery transluminal injury. Rapamycin treatment was started 2 days before injury and maintained for 2 weeks (1 mg/kg per 48 hours, ip). No significant differences in intima-to-media ratio were found between WT (1.1±0.1) and p27−/− mice (1.0±0.1) 4 weeks after injury. Rapamycin significantly (p< 0.05) reduced intima-to-media ratios in both WT (0.7±0.1) and p27−/− mice (0.5±0.1), compared with untreated mice. p27 deficiency did not alter the arterial wall proliferative response to injury. The inhibitory effect of rapamycin on intimal hyperplasia occurred via a p27-independent mechanism. The in vitro data showed that this effect was mediated through decreased proliferation and enhanced apoptosis.