Previous micropuncture studies on protein handling along the nephron could not exclude the possibility of contamination by extratubular proteins. Thus we developed a fractional micropuncture method. Renal tubules were punctured with an outer puncture pipette, into which an inner collection pipette was inserted repeatedly to collect tubular fluid, usually up to four fractions. The albumin concentration of tubular fluid was highest in the first fraction and gradually decreased to a constant level, indicating physiological albumin concentrations. On the other hand, low-molecular-weight protein (LMWP) concentrations showed no significant difference among the four fractions. By plotting the protein delivery in the fourth fraction along the nephron, glomerular filtrated protein concentrations were estimated by extrapolating the tubular fluid-to-plasma inulin concentration ratio into one. The glomerular filtrated albumin was 22.9 micrograms/ml (0.00062 in filtration coefficient), and that of LMWP was 72.1 (0.988). Albumin was almost evenly reabsorbed in early (37%) and late (34%) proximal convoluted tubules and the straight tubules (23%). On the other hand, LMWP was more strongly reabsorbed in the early proximal convoluted tubules (54%) than in the late ones (28%) or the straight portion (5%). The fractional micropuncture procedure provides direct evidence of protein handling along the nephron without extratubular protein contamination.