The chromosomal localization of c-myc sequences was determined by in situ hybridization in HL-60 cells (HL-60a) which contain an amplified c-myc locus and in an HL-60 subline (T-HL60) which has lost the amplification and has proportionately lower levels of c-myc RNA. While in HL-60a cells amplified c-myc sequences were found on the M3q+ marker chromosome, in T-HL60 cells one or few residual c-myc copies were found on a novel 4q+ marker chromosome. Comparative phenotypic analysis of HL-60a and T-HL60 cells show that the decrease in c-myc amplification/expression is not accompanied by changes in the malignant phenotype, namely in doubling time and clonogenic capability in semi-solid media. The significance of these results is discussed in the context of the role of c-myc amplification in the establishment and/or maintenance of the leukemic phenotype in HL-60 cells. In general, these results further underscore the utility of in situ hybridization analysis in identifying oncogene translocations which are not detectable by conventional karyotypic analysis.