Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3'-phosphate 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5'-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp -84 and bp -124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.