Phosphorothioate oligodeoxynucleotides (S-ODNs) have potential as anti-viral agents and are being investigated for the chemotherapy of AIDS. A high-performance liquid chromatographic method is described for the analysis, in urine and plasma, of a 28-unit deoxycytidine homopolymer (S-dC28) and a 28-unit S-ODN "antisense" to the rev gene of the human immunodeficiency virus. This method employs ion-pairing HPLC with a polymeric column. Tetrabutylammonium is used as the ion-pairing agent in a mobile phase of acetonitrile in pH 7.0 phosphate buffer. Analysis of the S-ODNs is relatively rapid (20 min) and sensitive (20 nm) and is accomplished by a gradient elution (22.5-30.0% acetonitrile) followed by ultraviolet (266 or 272 nm) absorption detection. This method is likely applicable, with appropriate modifications, to all S-ODNs of similar molecular weight regardless of sequence. The S-ODNs bind very strongly to plasma proteins but are readily prepared for analysis by a phenol extraction procedure. In a preliminary pharmacokinetic study in mice with S-dC28, very rapid elimination of the oligomer from plasma was observed (half-time, 11.6 min). Estimates for the apparent volume of distribution and total body clearance were 3 ml and 0.2 ml/min, respectively. It appears that the majority of the oligomer is eliminated by renal clearance (glomerular filtration), a property likely shared by all S-ODNs of similar molecular mass.