Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ER-positive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGF-stimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.