We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.