A suspension of cortical tissue fragments prepared by collagenase digestion of renal cortex obtained from fed and chronically acidotic (NH4Cl) rats was separated into four bands on a Percoll density gradient. By microscopic examination, vital staining with trypan blue, and histologic staining technique (periodic acid-Schiff) the F4 band was shown to contain only (greater than 98%) proximal tubules, whereas the F1 band was significantly enriched (70%) with distal tubules contaminated by glomeruli and short segments of proximal tubules. Intra/extracellular ratios for PAH of 15 were measured in the F4 band and of 2 in F1 band. ATP was 1.4 and 2.8 mumol/g in the F4 and F1 bands, respectively, and was stable for at least 60 min. The proximal F4 band was shown to be gluconeogenic (L-glutamine or L-lactate 2.5 mM as substrate) and to adapt to metabolic acidosis. The distal F1 band was shown to be glycolytic (glucose 2.5 mM) with no changes with acid-base status. All fractions were shown to metabolize glutamine, but the metabolic fate of this amino acid was different in proximal and distal structures. A F4/F1 activity ratio for the proximal cytoplasmic phosphoenolpyruvate carboxykinase enzyme of 2.6 and 4.3 was observed in normal and acidotic rats, respectively. In contrast, a F4/F1 ratio of 0.13 and 0.22 was observed for the distal cytoplasmic hexokinase enzyme. This preparation, therefore, allows the metabolism of a homogeneous population of proximal tubular fragments to be studied and can be used to obtain information on enzyme location within the nephron.