A calcium binding protein (CaBP) with an apparent relative molecular weight of 28,000 was localized in the kidney of Anolis carolinensis with antisera directed against vitamin D-dependent CaBP from either rat kidney (RRCaBP) or chick intestine (CICaBP). When extracts of female saurian kidneys were fractionated by gel filtration on Sephadex G-100, both calcium binding activity, as measured by the chelex resin assay, and CaBP immunoreactivity, as measured by radioimmunoassay for RRCaBP, were observed near the 28,000-Da region similar to RRCaBP and CICaBP. Utilization of the immunoblot technique following SDS-polyacrylamide slab gel electrophoresis resulted in cross-reactivity for CaBP in the Mr 28,000 region for both rat and anolian kidneys. Immunoreactive CaBP was localized in the nephron using the unlabeled antibody peroxidase-antiperoxidase technique. Distal tubules (DT) gave a strong, specific reaction with either antiserum, but not all cells of the DT reacted with equal intensity. Cells in the transition zone between the DT and the terminal tubules, and cells in the collecting tubules, were occasionally positive. Renal corpuscles, proximal tubules, and thin segments gave no specific localization of CaBP. The sexual segment of male kidneys was also negative. The results indicate that a calcium binding protein with an apparent molecular weight of 28K is highly conserved during vertebrate evolution, and thus may have widespread but specific functional significance. The localization of CaBP to the DT suggests that this protein may be involved in the selective reabsorption and/or excretion of calcium.