Amplification of sequences derived from 12q13-15 is frequent in human sarcomas and brain tumors. Detailed mapping studies of the amplified region are necessary for definition of the impact of these amplification events on the tumor cell phenotype. By using the genes in this region and genomic fragments isolated by chromosome microdissection, we have established a series of ordered probes from 12q13-15 for fluorescence in situ hybridization (FISH) and Southern blot analysis. These probes have been used for physical mapping of two portions of the interval from GLI to D12S8. The centromeric region extends 1.8 Mb from GLI to microclone M79 and contains at least five genes, including the cyclin-dependent kinase gene CDK4. The more telomeric region includes the p53 regulator MDM2 and covers 1.1 Mb. We used the same group of probes to determine the pattern of amplification in three cell lines and three tumor specimens carrying amplified sequences from 12q13-15. In addition, we used a yeast artificial chromosome (YAC) contig of several megabases covering the entire region from SAS to D12S8 for FISH to determine the pattern of amplification in the neuroblastoma cell line NGP-127. The results suggest that the MDM2 and CDK4 regions may be either coamplified or amplified independently, and they illustrate how the map positions of genes and their functions may interact to determine the pattern of DNA amplification in human malignancies.