The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.