The disposition of drugs may differ between pregnant and nonpregnant animals, necessitating a change in dosage. We hypothesized that volume of distribution or clearance may be different for aminoglycoside antibiotics in pregnant mares vs. nonpregnant lactating mares. To examine this hypothesis, we administered gentamicin sulfate to seven Thoroughbred and Quarterhorse mares on two occasions, followed by plasma drug gentamicin assay and pharmacokinetic analysis. The first dose was administered 1–4 weeks before parturition (mean weight 578 kg) and the second dose was administered in the period 1–4 weeks after parturition (mean weight 518 kg). The dose administered at each time was approximately 6.6 mg/kg, intravenously (i.v.). Plasma gentamicin concentrations were determined using fluorescence polarization immunoassay and pharmacokinetic analysis was performed using a two-compartment open model. The plasma concentration vs. time profiles and total area-under-the-curve were almost identical for mares at late gestation vs. early lactation. Mean volume of distribution at steady-state was 0.15 (±0.02) and 0.16 (±0.03) L/kg, systemic clearance was 1.06 (±0.17) and 1.11 (±0.17) mL/kg/min, and mean (harmonic) elimination half-life was 2.2 and 2.1 h, for pregnant and nonpregnant mares, respectively. We concluded that there were no differences in drug distribution and clearance between pregnant and nonpregnant lactating mares. Gentamicin was also assayed in plasma of newborn foals after an injection of 6.6 mg/kg to three of the mares within 60 min of parturition. Gentamicin was undetectable in plasma samples from these foals and, therefore, apparently does not cross the placenta of mares at term.