The purpose of this study was to compare the diagnostic performance of an immunoassay for placental alpha microglobulin-1 (PAMG-1) in vaginal fluid with the intra-amniotic instillation of indigo carmine for the diagnosis of membrane rupture in patients with an equivocal status.A prospective multi-site study was performed involving women reporting signs, symptoms, or complaints suggestive of rupture of membrane (ROM) between 21 and 42 weeks of gestation without obvious leakage of fluid from the cervical os during sterile speculum examination and without confirmation of ROM by traditional methods.A total of 140 patients were recruited with a prevalence of ROM of 19.3%. The PAMG-1 test had a sensitivity of 100.0% [confidence interval (CI) 0.87-1.0], specificity of 99.1% [(CI) 0.95-0.99], positive predictive value of 96.3% [(CI) 0.82-0.99], negative predictive value of 100.0% [(CI) 0.97-1.0], and ± likelihood ratios of 74.6 [(CI) 20.31-274.51] and 0.0 [(CI) 0.00-0.98].The PAMG-1 immunoassay in vaginal fluid yielded results that were comparable to those of the instillation of indigo carmine into the amniotic cavity; therefore, we propose that PAMG-1 is a sensitive and specific test to assess ROM in patients with an equivocal diagnosis based on simple tests. This finding prompts consideration for the use of the noninvasive PAMG-1 test in situations where the use of the invasive dye test is not practical.