Comamonas testosteroni 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil, and may therefore play a significant role in the bioremediation of hormonally active substances in the environment. We previously reported the isolation of the 3alpha-HSD/CR gene (hsdA) from C. testosteroni and two repressor genes, repA and repB, for hsdA transcriptional and translational regulation, respectively. In this work, we found that the expression of 3alpha-HSD/CR is closely connected to the distance between two 10 bp operator elements (OP1 and OP2) to which the RepA protein binds and therefore blocks the transcription of hsdA. The two 10 bp palindromic operator sequences are located upstream of hsdA (at 0.935 kb and at 2.568 kb on an EcoRI fragment) and are separated by 1.644 kb. In order to elucidate how the distance between OP1 and OP2 influences the repression of hsdA expression, we used E. coli cells transformed with plasmids carrying a set of deletions between OP1 and OP2. Our theory that a 'loop-structure' between the two operators is formed, which strongly influences hsdA transcriptional regulation, was proved by the increasing amounts of 3alpha-HSD/CR expression when the distance between the operators was too small to form the 'loop' structure. Moreover, additional -3, -5 and -7 nt deletions in each construct, known to result in DNA rotations and leading to altered orientations between the two operator sequences, reveal strong influences on hsdA expression the shorter the distance between the operators was. Our results demonstrate that a cis-regulating 'stem-loop' operator system is an important determinant in the regulation of the 3alpha-HSD/CR gene in Comamonas testosteroni.