Amplification of the erbB/EGF receptor and a structurally related gene, designated erbB‐2, have previously been detected in a variety of human tumors. In a series of human mammary tumor cell lines, analysis of transcripts of these genes revealed elevated levels of one or the other in more than 60% of tumors analyzed. Eight cell lines demonstrated erbB‐2 mRNA levels ranging from 4‐ to 128‐fold above those of normal controls. erbB‐2 expression was evaluated in comparison to the expression level of actin observed in these cell lines. There was no evidence of an aberrantly sized erbB‐2 transcript in any of these lines. Immunoblot analysis indicated elevation in levels of the 185‐kd product of the erbB‐2 gene expressed by these cells. In four lines erbB‐2 gene amplification in the absence of an apparent gene rearrangement was demonstrated. In a representative cell line of this type, SK‐BR‐3, the amplified erbB‐2 gene copies were located in an aberrant chromosomal location. Four additional cell lines, which demonstrated 4‐ to 8‐fold overexpression of erbB‐2 mRNA, did not exhibit gene amplification. In a representative cell line of this type ZR‐75‐1, an apparently normal chromosomal location was found for the erbB‐2 gene. Our findings indicate that overexpression of the erbB‐2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities.