We have reported that preinjection of zinc could ameliorate gentamicin-induced nephrotoxicity in vivo via the induction of metallothionein. The present project was designed to study whether preinjection of zinc could protect from the harmful effects of gentamicin on DNA synthesis, Na+-K+-ATPase activity, and free radical production in rat renal proximal tubules (PT) in vitro. PT were prepared and cultured from normal, saline-preinjected, and Zn-preinjected (Zn 10 mg/k/day s.c. 5 days) Wistar male rats weighing 80–120 g. The results showed that DNA synthesis and Na+-K+-ATPase activity were significantly suppressed in the normal and saline-preinjected rats' PT by addition of gentamicin to the media in the concentration of 12.4 mg/mL; however, these reactions were not suppressed by gentamicin in Zn-preinjected rats' PT, and malondialdehyde and hydroxyl radical production in Zn-preinjected rats' PT were significantly lower than those in the normal and saline-preinjected rats' PT (p<. 01). On the other hand, the metallothionein in Zn-preinjected rats' PT was very significantly higher than that in the normal and saline-preinjected rats' PT (p<. 001). Our data indicate that gentamicin-induced suppression of DNA synthesis and Na+-K+-ATPase activity in rat PT, which lead to renal injury, may be relevant to free radicals generated by gentamicin; and that preinjection of zinc could ameliorate gentamicin-induced nephrotoxicity via the induction of the metallothionein synthesis in rat PT to scavenge free radicals generated by gentamicin.