Uveal melanoma is the most common form of primary eye cancer. Monosomy 3, which is an unusual finding in tumors but is present in approximately 50% of uveal melanomas, is significantly correlated with metastatic disease. To obtain positional information on putative tumor suppressor genes on this chromosome, we have investigated tumors from 333 patients by comparative genomic hybridization, microsatellite analysis, or conventional karyotype analysis. A partial deletion of the long arm was found in eight tumors, and the smallest region of deletion overlap (SRO) spans 3q24-q26. We found six tumors with a partial deletion of the short arm and were able to define a second SRO of about 2.5 Mb in 3p25. This SRO does not overlap with the VHL gene. Our finding suggests a role for two tumor suppressor genes in metastasizing uveal melanoma and may explain the loss of an entire chromosome 3 in these tumors.