The novel mouse serine-threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides and growth factors via a phosphorylation-dependent mechanism requiring protein kinase C (PKC) activity. Structural comparison of the PKD catalytic domain with other kinases reveals a close similarity with MEK family kinases, which are activated upon phosphorylation of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in which putative activation loop serine residues 744 and 748 were mutated to either alanine or glutamic acid into COS-7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form upon phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid replacement mutants were partially activated. In vivo 32P-labeling and two-dimensional phosphopeptide mapping of PKD and catalytically inactive PKD mutants at serine 744, 748 or at both residues revealed that phorbol ester-sensitive phosphopeptides could be selectively eliminated from patterns observed as a result of these mutations. Treatment of cells with the PKC inhibitor GFI also prevented the appearance of phosphopeptide spots occuring in response to phorbol ester stimulation. These results provide direct evidence that PKD becomes activated in vivo as a consequence of PKC-mediated phosphorylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.