Abstract Positional cloning of the cancer-associated 20q13.2 amplicon identified two genes that display high mRNA levels in breast tumors and here we report the initial characterization of one of these gene products, designated Novel Amplified in Breast Cancer-1 (NABC1). Analysis of the primary structure of the NABC1 protein uncovered two regions of this protein with a high likelihood of forming coiled-coils and assembly of a mouse NABC1 cDNA showed that this protein is conserved between mouse and man. NABC1 antisera showed that, like its transcript, breast tumor lines that harbor amplification of 20q13.2 display high levels of the NABC1 protein compared to normal human fibroblasts or a breast cancer line that does not overexpress the NABC1 mRNA. Further, we conclude from studies using in vivo and in vitro approaches that the NABC1 protein forms detergent stable homodimers, and it is this homodimeric form that accumulates in cells that overexpress this protein. NABC1 mRNA exhibits a limited expression pattern in human tissue with high relative transcript levels observed only in brain and prostate. Immunofluorescence microscopy indicates NABC1 displays a punctate localization pattern in the cytoplasm of cultured cells, but biochemical fractionation indicates that this protein is not an integral component of membranous cytoplasmic organelles. Finally, overexpression of human NABC1 in mouse NIH/3T3 cells did not affect either the growth rate or anchorage-dependent growth properties, suggesting that NABC1 is not a prototypical oncogene.