We previously reported that prolactin-mediated macromolecular synthesis and mitogenesis are coupled to the activation of mitogen-activated protein kinase (MAPK) and p70 S6-kinase (p70S6K). Full activation of MAPK requires tyrosine and threonine phosphorylation whereas that of p70S6K requires serine phosphorylation. In the present study, okadaic acid, which inhibits serine/threonine protein phosphatase activity, was used to explore the linkage of MAPK and p70S6K activation to downstream effects of prolactin in Nb2 cells. The results show that 1 nM okadaic acid augmented prolactin-stimulated mitogenesis and synthesis of protein and DNA 250%, 42%, and 70%, respectively. Addition of okadaic acid alone a) stimulated and sustained p70S6K activity (5- to 8-fold) and MAPK (3.5- to 5-fold); and b) increased protein synthesis with the maximum effect being about equal to that of prolactin (2.1-fold with 1 nM okadaic acid versus 2.3-fold with 0.2 nM prolactin). However, okadaic acid did not affect DNA synthesis or mitogenesis. These results indicate that the activation of MAPK and p70S6K is necessary for stimulation of protein synthesis but not sufficient for prolactin-driven mitogenesis.