Probe substrates are an important tool for activity monitoring of human drug metabolizing enzymes such as cytochromes P450 (CYPs). In the present study we have tested human CYPs for metabolization of five proluciferin ester substrates which had previously only been known to be hydroxylated by CYP26A1. It was found that these substrates were converted by another 21 human CYPs, which belong to the CYP families 1 to 4, 7, and 26. Thus, 66 new pairs of enzyme and substrate were identified. Correlation analysis indicated the presence of three distinct sets of enzymes with high similarity in their activity profiles that encompass a total of 16 individual enzymes. Some of these newly identified correlations may serve as a starting point for further study of those human CYPs whose activities are not yet satisfactorily understood.