A novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2.8.2.22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (M(r) 290,000) is composed of four subunits (M(r) 70,000). The best donor substrate was 4-methylumbelliferyl sulfate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. The best acceptor substrate was alpha-naphthol, followed by phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.095 and 0.71 mM, respectively. One of the reaction products, p-nitrophenol inhibited the enzyme noncompetitively with respect to PNS, but competitively with respect to alpha-naphthol. The Ki values of PNP for PNS and alpha-naphthol were 0.89 and 0.12 mM, respectively. The other reaction product, alpha-naphthyl sulfate, inhibited the enzyme competitively with respect to PNS, but non-competitively with respect to alpha-naphthol. The Ki values of alpha-naphthyl sulfate for PNS and for alpha-naphthol were 2.72 and 1.7 mM. These results suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism.