Acquired insensitivity to the nephrotoxic effects of gentamicin develops in Fischer 344 rats after 10 to 14 days' treatment after development of histologic acute tubular necrosis in a setting of extensive histologic regeneration. To determine the relative importance of aminoglycoside exposure, necrosis, and regeneration in the induction of insensitivity, we examined the effect on gentamicin toxicity of prior non-aminoglycoside-mediated tubular necrosis, antecedent nonnecrotizing aminoglycoside exposure, and unilateral Nx-induced renal tubular hyperplasia. Pretreatment with potassium dichromate, which causes tubular necrosis in the same part of the renal cortex as gentamicin, reduced gentamicin-mediated elevation of Scr but had little effect on gentamicin-related tubular dysfunction or structural damage. Pretreatment with netilmicin, which does not cause tubular necrosis, increased the sensitivity of the kidney to gentamicin; toxicity occurred earlier and was more severe. Antecedent unilateral Nx had no demonstrable effect on susceptibility to gentamicin-associated dysfunction, but histologic renal tubular epithelial regeneration and recovery from dysfunction occurred earlier, These results suggest that necrosis and/or regeneration is the major prerequisite for development of gentamicin insensitivity and that the onset of insensitivity is temporally related to the appearance of necrosis and regeneration. However, non-aminoglycoside-mediated necrosis and regeneration fail to fully-re-create insensitivity, suggesting that exposure to gentamicin is also necessary.