A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.