There is currently no information available as to whether different renal fibroblast subpopulation can be identified and whether they show differences in functional properties. We therefore compared the growth characteristics of interstitial fibroblasts derived from the rabbit renal cortex and inner medulla (papilla) and sought cell-specific markers for the two populations of cells. Analyses of the population dynamics revealed that the mitotic lifespan of papillary fibroblasts (PF) is approximately 50% longer than that of cortical fibroblasts (CF), with the former going through 20 cumulative population doublings (CPD) before transition into terminally differentiated postmitotic cells compared with 9 CPD in CF. PF and CF populations contained three types of mitotically active cells (MFI, MFII, MFIII) and three types of postmitotic cells (PMFIV, PMFV, PMFVI) differentiating along a terminal cell lineage from MFI through PMFVI. In both PF and CF cultures the percent of MF-type cells decreased and the percent of postmitotic cells increased with successive doublings. Two-dimensional polyacrylamide gel electrophoresis of uniform clonal populations of MFIII-type cells revealed two specific proteins for PF-MFIII-type cells, pf1 and pf2, and three specific proteins for CF-MFIII-type cells, cf1, cf2, and cf3. Additionally, a monoclonal antibody was raised that does not recognize CF in culture, but reacts strongly with PF. These studies demonstrate that rabbit renal PF have a pattern of growth in vitro that is distinct from that of CF and that they can be positively identified by specific immunological and protein markers in vitro.