Protein tyrosine kinases (PTKs) have been implicated in signal transduction in a variety of cell types. B lymphocytes express the genes encoding for eight members of the src family of nonreceptor PTKs. Four of these PTKs (p55hlk, p53/56lyn, p59lyn, and p56lck) are activated by the ligation of mIg receptors. The functional roles of these PTKs in membrane-bound immunoglobulins (mIg) receptor-mediated activation of resting B lymphocytes were examined using the PTK inhibitor, herbimycin A. Here we show that mIg receptor-mediated B-cell proliferation and differentiation were inhibited by treatment with herbimycin A, while inhibitor-treated B cells retained LPS (mitogen) responsiveness for proliferation and antibody formation. Further studies demonstrated that herbimycin A blocked the G0 to G1 transition during B-cell activation. When the effects of herbimycin A were directly examined by a kinase activity assay, the enzymatic activity of each PTK was inhibited to varying degrees. The inhibition of PTK activity was also reflected by reduced tyrosine phosphorylation of intracellular substrates, including phospholipase C-γ. These results implicate PTK-dependent signaling pathways in the mIg receptor-mediated functional activation of B lymphocytes.