This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)(2)cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)(2)cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE(2), promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE(2)-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.