Transforming growth factor-alpha (TGF-alpha) secretion in the urine may reflect autonomous production by neoplasms. Previous measurements of TGF-alpha in the urine were limited by the large volumes of urine required for detection. Sensitive assays are required to detect TGF-alpha in small urine samples from individual patients with melanoma. An assay which detects ng quantities of immunoreactive TGF-alpha in 10 ml of urine using dialysis, reverse-phase high-performance liquid chromatography (HPLC), and Western blot of the HPLC fractions was used to study the urine from 6 melanoma patients and 6 normal human volunteers. No TGF-alpha was detectable in the first voided urine specimens from the normal volunteers. In contrast, TGF-alpha was detected in the urine specimens of two of three patients with primary melanomas and two of three patients with metastatic melanoma. The sensitivity and reliability of the assay were most affected by methods of urine collection, sample preparation, and storage. TGF-alpha was less often found in randomly voided urine samples from melanoma patients (1 of 3 detectable) as compared to first voided morning urine samples (3 of 3 detectable). This data indicates urinary TGF-alpha assay may be useful in the diagnosis and management of patients with melanoma.