Abstract:  There have been few reports of successful long‐term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long‐term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)^+/− mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long‐term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique.