Mutation spectrum of the retinoblastoma gene in osteosarcomas. We have performed an intensive mutation survey of the Rb gene in 63 osteosarcomas. Loss of heterozygosity (LOH) at the Rb locus was analyzed by using polymerase chain reaction at four intronic polymorphic sites, and 62.9% (39 of 62) of tumors showed LOH. Mutation analysis of the Rb gene was performed by Southern blot for structural anomalies, polymerase chain reaction-single strand conformation polymorphism analysis followed by direct genomic sequencing for subtle mutations, and immunohistochemistry for protein expression. The frequencies of each type of abnormalities were: structural anomalies, 28.6% (18 of 63); subtle mutations, 6.0% (3 of 50); negative protein expression, 53.6% (30 of 56); 54.5% (18 of 33) of tumors with LOH at the Rb locus were proved to show negative Rb expression, while 50.0% (11 of 22) of tumors without LOH also showed negative Rb, indicating that LOH at the Rb locus in osteosarcoma will not necessarily correlate with the actual inactivation of the Rb gene at the protein level. Findings in primary tumors were correlated with clinical outcome, and the presence of LOH and DNA alterations of the Rb gene were proved to indicate poor prognosis.