The importance of electrostatic interactions between charged residues at the P3 position of substrates and the S3 subsite of the cysteine protease clostripain was investigated. For this purpose quantitative enzymatic hydrolysis studies using steady state kinetics have been carried out within a set of N alpha-protected synthetic dipeptide ester substrates with systematic changes of their charge in the P3 position. It was demonstrated that, in contrast to the former postulated second anionic S3 subsite, the lowest specificity was for the hydrolysis of the positively charged substrates. However, this effect was strongly dependent on the individual amino acid at P1. Furthermore, we investigated how far these P3-S3 interactions reflect on the S' subsite specificity via acyl transfers. Apart from the general weak influence of the charge at P3 on the deacylation kinetics, nucleophiles with proline at P'1 play an extraordinary role. Surprisingly, in contrast to the poor primary lysine specificity, acyl transfer using P1 lysine substrates does not affect the nucleophile efficiency found with the corresponding arginine substrates.