We have compared a new commercially available non-radioactive protein kinase C (PKC) activity assay based on the fluorescent [A9,10K11]glycogen synthase1-11 analogue C1-peptide with a classical radioactive assay based on myelin basic protein4-14 (MBP4-14) and other substrates. The C1-peptide had lower affinity for PKC from rat brain than substrates such as MBP4-14, [S25]PKC alpha 19-31, and [A9,10K11,12]glycogen synthase1-12. The sensitivity of the C1-peptide-based assay was considerably lower than that of the MBP4-14-based assay. The C1-peptide was readily degraded in an ATP-independent manner by crude and DEAE-column chromatography-purified cytosolic extracts from rat brain, rat kidney, SK-N-MC and L929 cells. In rat kidney this degradation was not prevented by many common protease inhibitors. Phenylsepharose column chromatography separated the C1-peptide degrading activity from PKC. We conclude that the C1-peptide-based fluorescent PKC assay is applicable to highly purified PKC preparations but has low sensitivity and is not applicable to crude extracts due to substrate degradation.