Insulin-like growth factor-I (IGF-I) potentiates epidermal growth factor (EGF)-stimulated placental lactogen (hPL) secretion by cultured placental cells. In this study we have examined the effects of EGF and IGF-I, alone or in combination, on the differentiation of placental cells in culture and correlated this differentiation with hPL secretion. Addition of EGF (10 ng/mL) or IGF-I (100 ng/mL) alone in serum-free medium was associated with enlargement of the BhCG-positive (differentiated cytotrophoblast marker) mononucleated trophoblast cells. Concomitant addition of IGF-I and EGF resulted in more marked enlargement of the BhCG-positive cells, aggregation of the enlarged cells, and early syncytiotrophoblast formation. Measurement of hPL in the medium revealed that the stimulatory effect (P less than 0.05) of EGF on hPL secretion by total placental cells was proportional to the stimulatory effect of EGF on cell differentiation, as revealed by the obliteration of its stimulatory effect on hPL by BhCG-positive cells. The augmented stimulatory effect of EGF on hPL secretion by all cells in the presence of IGF-I was decreased (P less than 0.05) when expressed per number of human chorionic gonadotropin (BhCG)-positive cells. The dose-response curves of hPL stimulation by IGF-I in the presence of EGF revealed that the decreased effect of IGF-I on hPL secretion by BhCG-positive-staining cells was due to the difference in magnitude (approximately 1.5 times) of stimulation of BhCG staining of and hPL secretion by the cells. These results extend our previous suggestion that IGF-I in placenta, in addition to its effect on hPL secretion, affects cell differentiation; however, the two effects may not be interdependent.