We have cloned human protein kinase Bgamma (PKBgamma) and found that it contains two regulatory phosphorylation sites, Thr305 and Ser472, which correspond to Thr308 and Ser473 of PKBalpha. Thus it differs significantly from the previously published rat PKBgamma. We have also isolated a similar clone from a mouse cDNA library. In human tissues, PKBgamma is widely expressed as two transcripts. A mutational analysis of the two regulatory sites of human PKBgamma showed that phosphorylation of both sites, occurring in a phosphoinositide 3-kinase-dependent manner, is required for full activity. Our results suggest that the two phosphorylation sites act in concert to produce full activation of PKBgamma, similar to PKBalpha. This contrasts with rat PKBgamma, which is thought to be regulated by 3-phosphoinositide-dependent protein kinase 1 alone.