T4 decreases gentamicin nephrotoxicity, but the mechanism is unknown. This study investigated whether T4 affects renal processing of gentamicin by examining renal cortical gentamicin accumulation and proximal tubular lysosomal volume after 48 h of gentamicin (30 mg/kg twice daily) in rats (GT4) that had previously received 10 days of T4 (10 micrograms/100 g body wt). The pair-fed control group received only gentamicin. A T4-treated control group and an untreated control group were also pair fed and studied. At the end of the 12-day study the four groups did not differ in food intake, body weight, urine volume, urinary Na+ excretion, or urinary K+ excretion. Plasma creatinine, inulin clearance, and serum clearance of gentamicin were not different among the groups. Gentamicin accumulation was higher in G vs. GT4 (2.1 +/- 0.1 vs. 1.7 +/- 0.1 micrograms/mg protein, P less than 0.01). Blinded, computer-assisted electron microscopic analysis of outer cortical proximal tubules showed that lysosomes occupied a significantly smaller fraction of the cell volume in GT4 (8.1 +/- 0.5 vs. 11.6 +/- 1.1%, P less than 0.02). Thyroxine had no effect on lysosomal volume in non-gentamicin-treated rats. Except for scatter foci of apical vacuolization, proximal tubular cells of both groups were otherwise normal. The results show that chronic T4 administration decreases renal tubular accumulation of gentamicin, and this effect may in part explain its protective effect against gentamicin nephrotoxicity.