Recent studies have suggested that prostaglandin E₂ (PGE₂) subtype receptors (EP) are involved in cellular proliferation and tumor development. We studied the role of EP₁ and EP₄ PGE₂ subtype receptor antagonists AH-6809 and AH-23848B, respectively, in serum-induced 3T6 fibroblast proliferation. This was significantly reduced in a dose-dependent manner (IC₅₀ ~100 and ~30 µM, respectively) to an almost complete inhibition, without any cytotoxic effect. However, the effect of each antagonist on 3T6 cell cycle progression clearly differed. Whereas the EP₁ antagonist increased the G₀/G₁ population, the EP₄ antagonist brought about an accumulation of cells in early S phase. These effects were associated with a decrease in cyclin D and E levels in AH-6809-treated 3T6 cells and lower cyclin A levels in AH-23848B-treated fibroblasts with respect to control cells. The G₀/G₁ accumulation caused by AH-6809 seems to be intracellular Ca²⁺ concentration ([Ca²⁺]_i) dependent, because a 6-h 1 µM thapsigargin treatment allowed G₀/G₁-arrested cells to enter S phase. Similarly, treatment with 20 µM forskolin for 6 h allowed S-phase and G₂/M progression of AH-23848B-treated cells. This study shows that the inhibitory effect of the EP₁ and EP₄ antagonists on serum-induced 3T6 fibroblast growth is due to their effect at various levels of the cell cycle machinery, suggesting that PGE₂ interaction with its different subtype receptors regulates progression through the cell cycle by modulating cAMP and [Ca²⁺]_i.