Trophoblast giant cell differentiation is characterized by endoreduplication and expression of members of the prolactin (PRL) gene family and can be simulated in vitro via manipulations of the Rcho-1 trophoblast cell line. The regulation of trophoblast cell proliferation and differentiation involves tyrosine protein kinase signaling pathways. Treatment of Rcho-1 trophoblast cells with tyrosine kinase inhibitors disrupted differentiation-dependent expression of members of the PRL gene family and cytoskeletal organization. Activated p60(c-src) p62(c-yes), and p53/56(lyn) were present in the Rcho-1 rat trophoblast cell line and in differentiated trophoblast cells isolated from the developing rat placenta. p60(c-src) and p62(c-yes) were active in proliferating and differentiating trophoblast cells. During proliferation, p62(c-yes) exhibited distinct associations with other phosphoproteins (34, 66, 76, and 150 kDa). p53/56(lyn) was activated only in differentiating trophoblast cells. p53/56(lyn) showed a differentiation-dependent accumulation in cytoskeletal and membrane fractions, whereas p60(c-src) levels were virtually invariant in both fractions. Expression patterns of csk, a negative regulator of Src family kinase activities, were not consistent with its involvement in the differentiation-dependent activation of p53/56(lyn); however, there was some indication of the participation of a tyrosine phosphatase in the regulation of p53/56(lyn). In conclusion, p60(c-src), p62(c-yes), and p53/56(lyn) patterns of activation in trophoblast cells are consistent with their involvement in the control of trophoblast cell proliferation and differentiation.