OBJECTIVE We evaluated the usefulness of fluorescence in situ hybridization in the treatment of patients with equivocal cytology. METHODS Fluorescence in situ hybridization was performed in residual urine from 124 patients with a cytological diagnosis of cell clusters (22), atypical findings (46) and suspicious findings (56) who had a same day cystoscopy result and bladder biopsy within 6 months of the cytology diagnosis. Urologists and fluorescence in situ hybridization technologists were blinded to the matching fluorescence in situ hybridization and cystoscopy results, respectively. RESULTS In conjunction with cystoscopy fluorescence in situ hybridization was significantly more sensitive than cystoscopy alone for detecting cancer (87% vs 67%, p <0.001) and muscle invasive cancer (94% vs 56%, p = 0.031). Of the 124 equivocal cytology specimens 58 (47%) were positive by fluorescence in situ hybridization. Of these patients 53 (91%) had subsequent evidence of carcinoma, including Ta tumors in 17, Tis in 13, T1 in 8 and T2 or greater in 15, on the first followup biopsy. Three of the 5 remaining patients with a positive fluorescence in situ hybridization result and negative first followup biopsy had evidence of cancer at a later date, including TxN+ disease in 2 and Tis in 1. A total of 66 specimens were diagnosed as negative by fluorescence in situ hybridization. Of these patients 34 (52%) had negative biopsy results, whereas the remaining 32 (48%) demonstrated bladder cancer, including Ta disease in 20, Tis in 8, T1 in 2 and T2+ in 2. Cystoscopy detected 21 of the 32 tumors (66%) not detected by fluorescence in situ hybridization, while fluorescence in situ hybridization detected 17 of the 28 (61%) not detected by cystoscopy. CONCLUSIONS Our data suggest that fluorescence in situ hybridization with cystoscopy can aid clinicians in the diagnosis of bladder cancer in patients with equivocal cytology.