Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)