Cytogenetic investigation of uveal melanoma (UM) has revealed that monosomy 3 is the most frequent karyotypic abnormality, present in approximately 60% of cases. We investigated a cohort of 41 cases of UM, 19 of which retained two apparently normal copies of chromosome 3. Investigation of loss of heterozygosity (LOH) status was undertaken in an attempt to detect subcytogenetic loss of genetic material in those cases with two copies of chromosome 3. DNA from peripheral blood lymphocytes and fresh frozen or paraffin-embedded tumor tissue from 19 patients was amplified by the polymerase chain reaction for polymorphic loci on chromosome 3, including dinucleotide repeats, a tetranucleotide repeat, and polymorphic restriction enzyme sites. Three tumors showed LOH at multiple informative loci on both short and long arms of chromosome 3. Two additional tumors showed localized LOH on 3q, which corresponded to large deletions seen by cytogenetic analysis. The remaining 16 tumors showed retention of heterozygosity at all informative loci. This study did not detect the presence of cryptic deletions but revealed instead complete chromosomal homozygosity or functional monosomy, which probably occurred by loss and then duplication of the remaining chromosome 3. The demonstration of acquired isodisomy (functional monosomy) in a subset of UM increases the percentage of cases with monosomy 3 and provides further evidence for a central role of chromosome 3 loss in the molecular pathogenesis of uveal melanoma.