The insulin receptor (IR) form hybrids with the closely related insulin-like growth factor-I (IGF-I) receptor (IGF-I-R). Because most human breast carcinomas overexpress both the IR and the IGF-I-R, we evaluated whether the insulin/IGF-I hybrid receptor (Hybrid-R) is also overexpressed in these tumors and what role it plays in breast cancer biology. Using specific ELISAs and Western blots, we measured Hybrid-R content and function in 8 human cultured breast cancer cell lines and 39 human breast cancer specimens. Hybrid-R content and function were also compared to the content and function of the IR and the IGF-I-R. Hybrid-R content exceeded the IGF-I-R content in >75% of breast cancer specimens and was directly related to the molar ratio of both the IR and IGF-I-R content, suggesting that Hybrid-R formation occurred by random assembly of IR and IGF-I-R half-receptors. Hybrid-Rs became tyrosine autophosphorylated when breast cancer cells were exposed to IGF-I but not when they were exposed to insulin. In cells with an elevated Hybrid-R content, Hybrid-R autophosphorylation in response to IGF-I exceeded IGF-I-R autophosphorylation, suggesting that most of the IGF-I effect occurred via the Hybrid-R. Furthermore, Hybrid-Rs mediated growth in response to IGF-I, as indicated by experiments with blocking antibodies to the IGF-I-R. These data indicated therefore that: ( a ) Hybrid-Rs are present and play a major role in mediating the IGF-I signal in breast cancer; ( b ) their expression is directly related to IR overexpression; and ( c ) potential therapies designed to block IGF-I actions in breast cancer must take into account the role of these Hybrid-Rs.