In a recent report (Cho ct al., Proc. Natl. Acad. Sci. USA 97, 835-840, 2000), we showed that cancer cells of various cell types secrete cAMP-dependent protein kinase (PKA) into the conditioned medium and that in the serum of cancer patients this extracellular PICA (ECPKA) is upregulated 10-fold as compared with normal serum. Here, we characterized the enzymatic properties of ECPKA that is present in the conditioned medium of PC3M prostate cancer cells and in the serum of cancer patients, and we compared ECPKA with PICA found in the cell extracts of PC3M cells. ECPKA present in the conditioned medium and human serum was not activated by cAMP addition, but intracellular PHA activity was totally dependent on the addition of cAMP. This indicates that the ECPKA is present in active, free C subunit form, whereas intracellular PICA is present in inactive holoenzyme form. ECPKA activity increased in a substrate concentration- and time-dependent manner, as did intracellular PICA Both ECPKA and intracellular PICA activities were specifically inhibited by the PICA inhibitor protein, PHI. However, ECPKA activity was more temperature-sensitive than intracellular PKA; after two cycles of freezing/thawing, only 20% of initial ECPKA activity was detected compared with over 40% of intracellular PICA activity. Western blot analysis revealed the presence of a 40 kDa C-alpha subunit of PICA in both conditioned medium and in the serum of cancer patients. These results suggest that ECPKA, out of the context of cAMP regulation, may function as a growth factor promoting cell growth and transformation; thus, it may serve as a tumor biomarker.