The variability of single nephron filtration fraction (SNFF) and the relationship between filtration fractions determined simultaneously from 125I albumin (SNFFALB) and hematocrit (SNFFHCT) values were assessed by multiple samplings, 5 to 8 per kidney, in normal, hydropenic, Munich-Wistar rats. Single nephron filtration rate (SNGFR) measurements served as an independent marker of internephron heterogeneity. Whether derived as SNFFALB or SNFFHCT, a wide degree of internephron variability in SNFF, comparable to that found for SNGFR (P greater than 0.2) was observed in all kidneys. Hematocrit-derived SNFF values, however, were some 80% higher than those obtained with 125I albumin (SNFFHCT 0.447 +/- SEM 0.016 versus SNFFALB 0.258 +/- 0.009, P less than 0.001). The disparity in results obtained by the two methods disappears if the efferent arteriolar hematocrit (Hctg) is assigned a value 17 +/- SEM 1.4% higher than the corresponding femoral arterial hematocrit and might well be a reflection of the perfusion of superficial glomeruli with erythrocyte rich blood produced by the "plasma-skimming" phenomenon. The data suggest that multiple estimates of SNGFR and SNFF in a given rat kidney are needed to provide representative mean values for glomerular dynamic studies.