We have identified a Ser/Thr kinase associated with the B cell receptor (BCR) complex as protein kinase C mu (PKC mu). PKC mu activity is up-regulated after cross-linking the BCR and CD19 on B cells, and PKC mu co-precipitates with Syk and phospholipase C-gamma 1/2 (PLC gamma 1/2). In vitro phosphorylation of fusion proteins showed that both Syk and PLC gamma 1 are potential substrates of PKC mu in vivo. Analysis of mutants of the chicken B cell line DT40 deficient in either Syk, Lyn, Btk, or PLC gamma 2 revealed that BCR-induced activation of PKC mu, like activation of PLC gamma 2, requires Syk and is partially regulated by Btk, but is Lyn independent. PKC mu can down-regulate the ability of Syk to phosphorylate PLC gamma 1 in vitro. Thus, PKC mu may function in a negative feedback loop regulating BCR-initiated signaling cascades.