A simple method for the configurational analysis of the four major 2'-deoxynucleoside 5'-[160, 180] phosphorothioates, (1), and adenosine 5'-[160, 180] phosphorothioate, (1a), is described. The method involves permethylation of (1,1a) with diazomethane or dimethyl sulphate, to generate the corresponding S-methyl-O-methyl phosphorothioate, (2,2a), 8P(3:1 DMF:d4-MeOH) +30ppm. Having assigned the diastereoisomers of (2,2a) to their corresponding 31P.n.m.r. resonances, the 18O-isotope is located by examination of 31P(180) isotope shifts. A larger shift, (0.05ppm), is observed on the diastereoisomer with 180 in the P-O position than in the P-OMe position, (0.02ppm). Configurations of (2,2a) were assigned by partial hydrolysis of the Sp diastereoisomer of a 1:1 mixture of the diastereoisomers of 2'-deoxyribonucleoside- or adenosine 5'-O-methyl phosphorothioate diester, (3,3a), 8P(3:1 DMF:d4-MeOH) +58ppm, by snake venom phosphodiesterase. Subsequent methylation of (3,3a) yielded correspondingly Sp-deficient samples of (2,2a). In all cases Sp(2,2a) resonates downfield of Rp(2,2a) by ca. 0.08ppm whilst Sp(3,3a) resonates upfield of Rp(3,3a) by ca. 0.12 ppm, and shows shorter retention time on reverse-phase h.p.l.c. The method is demonstrated by the stereochemical study of the hydrolysis of (3a) by bovine intestinal mucosa 5'-nucleotide phosphodiesterase, (EC 3.1.4.1), shown to proceed with overall retention of configuration at phosphorus, indicating the involvement of a covalent enzyme intermediate. Mung bean nuclease, (EC.3.1.30.1), is shown to hydrolyse 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl) phosphorothioate with inversion of configuration, consistent with a single step mechanism. The iodine-mediated desulphurisation of a dinucleoside phosphorothioate, Sp 5'-O-(2'-deoxyadenosyl)-3'-Q-thymidyl phosphorothioate, occurs with epimerisation at phosphorus in aqeous pyridine, but with 75% inversion of configuration in aqueous lutidine. These results implicate pyridine as a nucleophilic catalyst in the reaction, several displacements by pyridine causing loss of configuration at the phosphorus centre prior to displacement by water. Less nucleophilic lutidine shows reduced participation, allowing direct displacement of sulphur by water.