The N-methylthiotetrazole side chain (NMTT) that is present on several cephalosporins has been implicated in the development of antibiotic-associated hypoprothrombinemia. A randomized three-way crossover trial was conducted to compare the release of the NMTT side chain from three NMTT-containing antibiotics. Single 2-g doses of moxalactam, cefoperazone, and cefotetan were given, followed by serial blood and urine sampling. The concentrations of the parent compound and the NMTT side chain in plasma, urine, and the reconstituted antibiotic solution were determined by high-pressure liquid chromatography. Peak NMTT concentrations ranged from 0.42 to 16.50 μg/ml and were significantly higher after moxalactam administration than after cefoperazone or cefotetan administration (P < 0.01). The NMTT trough concentrations (12.5 h) ranged from nondetectable to 2.47 μg/ml and tended to be greater following cefoperazone administration. The amounts of NMTT administered (e.g., the amount in the reconstituted antibiotic solution) were 25.8 ± 1.4, 15.2 ± 0.9, and 22.1 ± 3.0 mg following moxalactam, cefoperazone, and cefotetan administration, respectively (P < 0.01). In contrast, urinary recoveries of NMTT were 57.4 ± 26.2, 73.6 ± 44.3, and 29.7 ± 22.9 mg following moxalactam, cefoperazone, and cefotetan, respectively. The amount of NMTT formed in vivo and excreted unchanged, as assessed by subtracting in vitro NMTT formation from NMTT urinary recovery, was significantly higher after cefoperazone than after moxalactam or cefotetan administration (P < 0.05). The discrepancy between in vitro NMTT production (moxalactam > cefotetan > cefoperazone) and the amount of NMTT followed in vivo and excreted unchanged (cefoperazone > moxalactam > cefotetan) demonstrated that the in vivo production of NMTT is dependent on the disposition of the parent cephalosporin.