By employing RT-PCR in conjunction with 3′-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3′,5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with β-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.