Clinically observed drug interactions with cytochrome p450 (p450) enzymes have increased the need to assess drug interactions of new chemical entities early in the discovery process. To meet this need, fluorogenic substrates have been commercialized. However, only limited evaluations of their utility and comparisons to drug probes have been reported. This study examines the correlation between IC50 values obtained with fluorogenic and conventional drug probes for structurally diverse inhibitors of the five major human p450 isoforms. In general, correlations are weak, with significant numbers of compounds being missed as inhibitors by either probe. For p450s 1A2, 2C9, and 2C19, correlation coefficients were above 0.6 with slopes that ranged from 1.5 to 4.2. However, for p450s 1A2 and 2C9, about 20% of compounds were not included because an IC50 value could not be determined with one of the two probes. CYP 2C19 had the highest correlation (correlation coefficient 0.84), with a slope of 2.0 and less than 5% of compounds excluded. CYP 2D6 showed a good correlation for IC50 values less than 10 microM. However, at higher IC50 values, a high degree of scatter was observed. CYP 3A4 had the weakest correlation, and a large number of compounds were excluded with the fluorogenic probe. Overall, the study shows the care needed when selecting fluorogenic probes and the caution needed when results with fluorogenic probes are used to drive structure-activity relationships with respect to drug interactions.