We previously determined that the urine of interstitial cystitis (IC) patients specifically contains a factor (antiproliferative factor [APF]) that inhibits primary bladder epithelial cell proliferation, and that it has significantly decreased levels of heparin-binding epidermal growth factor–like growth factor (HB-EGF) and increased levels of epidermal growth factor (EGF) compared with urine from asymptomatic controls and patients with bacterial cystitis. We sought to confirm the specificity of these findings for IC using a larger patient population, including control patients with a variety of urogenital disorders. Clean catch urine specimens were collected from 219 symptomatic IC patients, 113 asymptomatic controls without bladder disease, and 211 patients with various urogenital diseases including acute bacterial cystitis, vulvovaginitis, chronic nonbacterial prostatitis, overactive bladder, hematuria, stress incontinence, neurogenic bladder, benign prostatic hyperplasia, bladder or pelvic pain without voiding symptoms, bladder cancer, prostate cancer, or miscellaneous diagnoses including anatomic disorders. APF activity was determined by ³H-thymidine incorporation into primary normal adult human bladder epithelial cells. HB-EGF and EGF levels were determined by enzyme-linked immunosorbent assay. APF activity was present significantly more often in IC than control urine specimens (P <0.005 for IC vs any control group; sensitivity = 94%, specificity = 95%, P <10⁻⁸² for IC vs all controls). HB-EGF levels were also significantly lower and EGF levels significantly higher in IC urine than in specimens from controls (P <10⁻⁸⁴ and P <10⁻³⁶, respectively). These findings confirm the utility of APF, HB-EGF, and EGF as markers for IC. Understanding the reasons for altered levels of these markers may lead to understanding the pathogenesis of this disorder.