The purpose of this study was to evaluate the reliability of vaginal washing fluid creatinine level for the diagnosis of premature rupture of membranes (PROM). A prospective diagnostic study performed in Shahid Sedoughi Hospital on 160 pregnant women (30 definite PROM, 30 no PROM and 100 suspected PROM) at 28-40 weeks of gestation. The vagina was washed by injection with a syringe filled with 3 ml of saline solution, and the washing fluid was collected from the posterior vaginal fornix and send to laboratory. Creatinine values in vaginal washing were measured and compared. The mean vaginal fluid creatinine levels in definite PROM group, suspected PROM and no PROM were 0.40 +/- A 0.20, 0.16 +/- A 0.04 and 0.08 +/- A 0.01 mg/dl, respectively, where the difference was statistically significant (P = 0.001). The sensitivity, specificity, positive and negative predictivity values and accuracy were 98.7, 100, 100, 98.8 and 87.1%, respectively, in detecting PROM by evaluation of vaginal fluid creatinine concentration with cut-off value of 0.14 mg/dl. This study showed that creatinine determination in vaginal washing fluid is a useful marker for PROM diagnosis. It is a reliable, simple, cheap and rapid test.