We recently reported that in vivo phosphorylation of urokinase-type plasminogen activator on Ser138/303 prevents its catalytic-independent ability to promote myelomonocytic cell adherence and motility. We now show that Ca2+ activated, phospholipid-dependent protein kinase C from rat brain phosphorylates in vitro a peptide corresponding to prourokinase residues 133-143 (DGKKPSSPPEE) and the full-length molecule on Ser138/139. The in vivo involvement of the protein kinase C isoenzyme family is supported by the finding that inhibition of kinase C activity prevents prourokinase phosphorylation on Ser138/303 in A431 human carcinoma cells. Conversely, a short treatment of A431 cells with phorbol myristate acetate increases the extent of phosphorylated prourokinase and, concomitantly, affects its function; under these conditions, the capability of prourokinase to up-regulate U937 monocyte-like cell adherence is severely impaired, although receptor binding is unaltered. By the aid of a "phosphorylation-like" variant (Ser138 to Glu) we show that modification of Ser138 is sufficient to confer to prourokinase the antagonistic properties observed following in vivo stimulation of protein kinase C activity. These observations provide the first evidence that protein kinase C directs the formation of a receptor competitive antagonist by regulating the in vivo phosphorylation state of prourokinase.