D-Serine causes necrosis of S3 segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Co-injection of -amino-iso-butyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S3, thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. (1) We determined the fractional excretion (FE) of amino acids and glucose in rats after i.p. injection of D-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. (2) We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to about 30% of control. This decrease was prevented when equimolar GSH was co-injected with D-serine. (3) To find out why AIB protected the tubule from D-serine toxicity, we microinfused [¹⁴C]-D-serine or [¹⁴C]-AIB (0.36 mmol/l) together with ³H-inulin into late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of [¹⁴C]-D-serine and [¹⁴C]-AIB amounted to 55 and 70 %, respectively, and 80 mmol/l of AIB or D-serine, respectively, mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a ten-fold higher AIB concentration. We conclude from these results that (i) D-AAO-mediated D-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished; (ii) AIB prevents D-serine-induced tubulopathy by inhibition of D-serine uptake in S3 segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.