In 8 Holstein cows, 50 colony-forming units (CFU) Of Escherichia coli was administered into 1 mammary gland. Infections were established in all inoculated glands. In 4 of the 8 cows, 500 mg of gentamicin sulfate was administered by intramammary infusion 14 hours after inoculation; the other 4 cows were untreated controls. Infusions of gentamicin also were given after each of the 3 successive milkings after the initial infusion, so that a total dose of 2 g of gentamicin was given to each of the treated cows. During the 33-hour treatment period and for the first milking after the last infusion of gentamicin, the treated cows had a mean gentamicin concentration of greater than or equal to 31.0 microgram/ml in milk samples that were collected from inoculated quarters immediately before each milking. Concentrations of 0.34 and 0.69 microgram of gentamicin were detected in milk from 2 cows at 8 days after inoculation with E coli. Mean serum concentrations of gentamicin were > 0.37 microgram/ml throughout the treatment period and the first 12 hours after the last infusion, with a mean peak concentration of 0.96 microgram/ml at 24.4 hours. The range of peak concentration of gentamicin detected in urine from all treated cows was 42 to 74.4 microgram/ml. Peak concentration of E coli in milk in the treated cows (6.08 +/- 1.02 log10 CFU/ml) did not significantly (P > 0.05) differ from that of the control cows (5.26 +/- 1.00 log10 CFU/ml). Similarly, mean duration of infection in the treated cows (54 hours) did not differ significantly from that of the control cows (48 hours). The treatment groups also did not differ significantly in peak concentrations of albumin or IgG1 in milk, although mean concentrations of albumin and IgG1 at 16 hours after inoculation, and of albumin at 20 hours, was significantly (P < 0.05) higher in the milk from control cows than from the treated cows. Mean values of peak rectal temperature and of mean rectal temperature throughout the trial did not differ between the groups. At the end of the 4-week trial, 1 of 4 inoculated glands in treated cows and 3 of 4 in control cows had somatic cell counts less than or equal to preinoculation concentrations (5.18 log10 cells/ml). Intramammary administration of gentamicin did not affect the duration or severity of experimentally induced E coli mastitis. In addition, substantial concentrations of gentamicin were detected in the serum of treated cows, suggesting that intramammary treatment may result in prolonged drug residues in tissue.