New 4-phenylbutanoyl-2(S)-acylpyrrolidines and 4-phenylbutanoyl-L-prolyl-2(S)-acylpyrrolidines were synthesized. Their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. In the series of 4-phenylbutanoyl-2(S)-acylpyrrolidines, the cyclopentanecarbonyl and benzoyl derivatives were the best inhibitors having IC(50) values of 30 and 23 nM, respectively. This series of compounds shows that the P1 pyrrolidine ring, which is common in most POP inhibitors, can be replaced by either a cyclopentyl ring or a phenyl ring, causing only a slight decrease in the inhibitory activity. In the series of 4-phenylbutanoyl-L-prolyl-2(S)-acylpyrrolidines the cyclopentanecarbonyl and benzoyl derivatives were not as active as in the series of 4-phenylbutanoyl-2(S)-acylpyrrolidines. The hydroxyacetyl derivative did however show high inhibitory activity. This compound is structurally similar to JTP-4819, which is one of the most potent prolyl oligopeptidase inhibitors. The acyl group in the two series of new compounds seems to bind to different sites of the enzyme, since the second series of new compounds did not show the same cyclopentanecarbonyl or benzoyl specificity as the first series.