The influence of fixation on the immunocytochemical localization of tissue kallikrein in the kidney has been evaluated using both monoclonal and polyclonal antibodies. These studies have provided several results relevant to kallikrein localization in kidney: (1) the intensity and distribution of immunostaining with both polyclonal and monoclonal anti-kallikrein antibodies is fixation-dependent; (2) the most intense and consistent localizations of kallikrein are in the connecting tubule and the cortical collecting duct of the nephron; (3) kallikrein-like immunoreactivity is seen in proximal tubules with polyclonal but not with non-cross-reactive monoclonal antibodies; and (4) fixatives which disrupt membranes reveal a kallikrein-like antigen in straight tubules of the outer medulla. However, immunostaining with monoclonal antibodies indicates that much of the observed immunostaining at this site probably represents cross-reactivity with another member of the kallikrein family of enzymes.