Rolofylline is a potent, selective adenosine A1 receptor antagonist that was under development for the treatment of patients with acute decompensated heart failure and renal function impairment. This was a phase I, randomized, open-label, 2-period, fixed-sequence study in 19 healthy adult volunteers to examine the effect of multiple intravenous rolofylline doses on the single-dose pharmacokinetics of midazolam, a sensitive CYP3A4 substrate. In period 1, subjects received a single oral dose of midazolam 7.5 mg on day 1. In period 2, subjects received 30 mg, 4-hour infusions of rolofylline (intended clinical dose and duration) once daily for 4 consecutive days; midazolam 7.5 mg was coadministered on day 4. The geometric mean ratios and 90% confidence intervals for AUC0-infinity and Cmax of midazolam in the presence/absence of rolofylline were 1.20 (1.12-1.29) and 1.17 (1.03-1.32), respectively. The apparent terminal half-life (t1/2) for midazolam was similar in the presence/absence of rolofylline (4.31 and 4.27 hours, respectively). The geometric mean ratios (90% confidence intervals) for AUC0-infinity and Cmax of 1'-hydroxymidazolam in the presence/absence of rolofylline were 1.04 (0.96-1.13) and 0.98 (0.84-1.14), respectively. The t1/2 for 1'-hydroxymidazolam was slightly higher in the presence relative to absence of rolofylline (4.24 and 3.17 hours, respectively). Multiple doses of intravenous rolofylline 30 mg for 4 days were generally well tolerated and did not result in clinically important inhibition of CYP3A4 as indicated by little or no change in the pharmacokinetics of midazolam.