The properties of tubular glutarate uptake and the coupling to p-aminohippurate (PAH) transport were studied on isolated nonperfused S2 segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were totally collapsed, the tubular glutarate uptake may be assumed to represent the quantity transported across the basolateral membrane. The results show that the S2 segments effectively accumulated 14C glutarate (500 micron). The cell/bath 14C-glutarate concentration ratio reached maximum values of about 20 after a 20-min incubation period. The tubular 14C glutarate accumulation could be markedly depressed by lithium (5 mM) but not by probenecid (1 mM), which, however, inhibited tubular 3H-PAH (1 microM ) uptake. External PAH (0.1 mM) stimulated efflux of 14C-glutarate from S2 segments preloaded with 14C-glutarate (500 microM), and external glutarate stimulated tubular uptake of 3H-PAH (1 microM), providing evidence for glutarate-PAH countertransport in proximal S2 segments. The phorbol ester, phorbol 12-myristate 13-acetate (0.1 microM), did not affect steady-state cell/bath 14C-glutarate concentration ratio nor the initial 14C-glutarate transport rate. Protein kinase C may, therefore, not be a regulator of basolateral glutarate transport in renal S2 proximal tubules.