We sought a cultured cell line with Proline Oxidase activity to study the regulation and physiologic role of the enzyme in mammalian tissues. Among the cell lines tested, only LLC-RK1 cells, derived from rabbit kidney, had significant Proline Oxidase activity; the Km for proline of the enzyme from these cells was similar to that for the liver enzyme. LLC cells, Proline Oxidase positive, were able to convert proline to CO2. In contrast, CHL cells, Proline Oxidase negative, did not have this capability. The presence of Proline Oxidase in LLC cells and the absence of the enzyme in fibroblasts suggest that Proline Oxidase may serve as a marker enzyme for distinguishing parenchymal kidney cells from fibroblasts in culture. Cells transformed by SV40 virus and cells transformed by methylcholanthrene had activities higher that the parent cell line, but this effect of transformation could not be generalized to all transformed cells. Finally, L-hydroxy proline at 100-fold greater concentration than substrate L-proline failed to decrease proline oxidation. This finding suggests distinct degradative enzymes for these two amino acids.