Cefotetan, a new broad-spectrum 7 alpha-methoxycephalosporin antibiotic, was assayed in plasma and urine by means of reversed-phase high-performance liquid chromatography. Commercially available cefotetan exists in two epimeric forms. The procedure described allows the separation and quantitation of both epimers. For the first time a different pharmacokinetic behaviour (t1/2 = 3 h versus 4 h) for each epimer after intravenous injection to healthy volunteers is demonstrated. It is assumed that one epimer is bound to a greater extent to serum proteins and is therefore responsible for the differences observed. As both epimers exhibit similar antibacterial activity, it seems doubtful whether these differences would have clinical significance. Iothalamic acid was determined simultaneously as a marker of kidney function.