Intracellular localization of two molecular species of calpain (Ca2+-dependent cysteine proteinase) was studied by immunocyto- and histochemical methods employing antibodies strictly monospecific for the respective antigens. Apparent immunological cross-reactivity between the larger subunits of calpain I (low Ca2+-requiring form) and calpain II (high Ca2+-requiring form) was calculated to be 15-17%, and two steps of affinity chromatography were needed to obtain antibodies which can discriminate between the two proteases. Indirect immunofluorescent staining of cultured PK 15 cells revealed diffuse staining of the cytoplasm with both antibodies against calpain I and calpain II. Preincubation with Ca2+-ionophore had no effect on the staining patterns. Sections of porcine kidney were stained by the avidin-biotinylated peroxidase complex method. The proximal and distal tubules and collecting duct were stained, but the glomerulus, macula densa, and vascular vessels were not stained by either anti-calpain I or anti-calpain II antibodies.