In Escherichia coli as many as nine different genes coding for proteins with significant homology to peptidyl-prolyl cis/trans-isomerases (PPIases) have been found. However, for three of them, the histidine-rich SlyD, the homologous gene product of ORF149, and parvulin-like SurA, it was not known whether these proteins really possess PPIase activity. To gain access to the full set of PPIases in E. coli, SlyD, the N-terminal fragment of SlyD devoid of the histidine-rich region, as well as the protein product of ORF149 of E. coli named SlpA (SlyD-like protein) were cloned, overexpressed, and purified to apparent homogeneity. On the basis of the amino acid sequences, both proteins proved to be of the FK506-binding protein type of PPIases. Only when using trypsin instead of chymotrypsin as helper enzyme in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal fragment can be measured. For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were obtained. Surprisingly, the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region. Because the PPIase activity of SlpA could be established as well, we now know eight distinct PPIases with proven enzyme activity in E. coli.