Adriamycin-resistant HL-60 (HL-60/ADR) cells possess high constitutive levels of the proteolytically generated catalytic fragment of protein kinase C, M-kinase, resulting in the generation of pp130 in vitro. In this report, we have demonstrated the presence of a Ca2(+)-activated protease in extracts from HL-60/ADR cells, but not in wild-type cells, which resulted in the generation of 26 and 86 kDa phosphoproteins in vitro, as well as in the reduction of pp130. The formation of pp26 and pp86 and the reduction in pp130 were prevented by the serine/cysteine protease inhibitor, leupeptin. A 26 kDa phosphoprotein with a similar isoelectric point was also evident in both cell lines following metabolic labeling in vivo, although the phosphorylation of pp26 was much greater in resistant cells. These data suggest that the presence of Ca2(+)-dependent protease activity is a phenotypic characteristic of this multidrug resistant cell line.