The comparative pharmacokinetics and in vivo production of N-methylthiotetrazole (NMTT) were evaluated following administration of cefoperazone, cefotetan, and cefmetazole. In a randomized-crossover manner, 11 healthy male volunteers received single 2-g intravenous doses of each agent and serial blood and urine samples were collected. Concentrations of NMTT and the parent compound in plasma, urine, and the reconstituted antibiotic solution were determined by high-pressure liquid chromatography. The amounts of NMTT administered were 6.06 ± 0.46, 14.4 ± 0.87, and 17.4 ± 1.06 mg for cefoperazone, cefotetan, and cefmetazole, respectively (P < 0.05). The mean NMTT plasma concentration-time profiles following administration of each cephalosporin were markedly different. Six hours after dosing, NMTT concentrations in plasma following cefoperazone administration were higher than those following administration of cefmetazole and cefotetan. Urinary recoveries of NMTT averaged 137.0 ± 37.1, 38.3 ± 6.98, and 25.2 ± 5.95 mg following administration of cefoperazone, cefotetan, and cefmetazole, respectively (P < 0.01). The apparent amount of NMTT produced in vivo, calculated by subtracting the amount of NMTT administered from the amount of NMTT excreted in urine, was significantly lower following cefmetazole administration than after administration of cefoperazone and cefotetan (P < 0.01). The discrepancy between in vitro NMTT production (cefmetazole > cefotetan > cefoperazone) and the amount of NMTT formed in vivo and excreted unchanged (cefoperazone > cefotetan > cefmetazole) suggests that in vivo production of NMTT is dependent on the disposition of the parent cephalosporin. These results further suggest that cephalosporins which undergo extensive biliary excretion, such as cefoperazone, are associated with the greatest amount of in vivo release, whereas cephalosporins which are primarily renally excreted, such as cefmetazole, are associated with the lowest in vivo production of NMTT.