2,6-Dichloro-4-nitrophenol (DCNP)-35sulfate was identified and quantified by an HPLC-radiometric assay following its biosynthesis in vitro from 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAP35S) by phenolsulfotransferase (PST) of rat liver cytosol. Acid hydrolysis of DCNP-35sulfate produced almost stoichiometric release of inorganic 35sulfate and DCNP. In two-substrate experiments of sulfation of p-nitrophenol (p-NP) or dopamine (prototype substrates for P and M human PST forms), 10 microM DCNP inhibited the reactions by about 15 and 78%, respectively. This contrasts with its action on PST of human origin where the P-PST was more sensitive to DCNP inhibition. In all mixed bi-substrate experiments, a reciprocal relationship between the two sulfated products was observed. Kinetic data showed that p-NP inhibited the sulfation of DCNP competitively. Likewise the sulfation of p-NP and dopamine was competitively inhibited by DCNP. However, non-competitive inhibition was observed in the sulfation of p-NP by DCNP, measured at varying concentrations of PAP35S. The above kinetic data suggest that DCNP is an alternate-substrate inhibitor of rat liver PST.