A single spot urine collection to measure the ratio of 6 beta-hydroxycortisol (6 beta-OHC) to free cortisol (C) has been proposed as a research tool for the assessment of CYP3A4 induction. However, intraindividual variability in 6 beta-OHC/C under basal conditions and conditions of induction has not been prospectively evaluated, and findings on the correlation between morning spot and 24-hour urinary ratios have been conflicting. In this study, the variability in morning spot and 24-hour urinary 6 beta-OHC/C ratios was assessed in 15 healthy adult male volunteers before, during, and after oral administration of rifampin 600 mg once daily for 14 days. In addition, the correlation between morning spot and 24-hour urinary ratios measured under baseline, maximum induction, and postinduction was determined. Intraindividual coefficients of variation (CVs) at baseline for the morning spot and 24-hour ratios were 54.3% and 57.1%, respectively, and were not changed significantly during induction. No significant differences were detected in the variability between the morning spot and 24-hour ratios at baseline, maximum induction, or postinduction. A good correlation (r = 0.61, p < 0.0001) was detected between the mean morning spot and 24-hour urinary ratios. Mean (+/- SEM) percent increases in the morning spot and 24-hour ratios at maximum induction relative to baseline were 320% +/- 73% and 137% +/- 30%, respectively (p = 0.019). All 15 subjects had an increase in the mean morning spot ratio at maximum induction relative to baseline, whereas 12 subjects showed an increase in the mean 24-hour ratio. The time course of changes in the mean morning spot urinary ratio in response to a 14-day course of rifampin was also similar to that reported previously in a study using 24-hour urine collections. These findings suggest that measurement of the morning spot urinary 6 beta-OHC/C ratio is an effective and efficient method for evaluating the potential of investigational agents to induce CYP3A4.