We generated DNA expression vectors encoding the full-length neu cDNA (designated pNeu_N), the neu extracellular domain (pNeu_E), or the neu extracellular and transmembrane domains (pNeu_TM). The 293 cells transfected with pNeu_N or pNeu_TM expressed the neu extracellular domain on the surface membrane, whereas 293 cells transfected with pNeu_E secreted the extracellular domain of neu into the culture supernatant. We examined whether i.m. injection of either of these plasmids could induce protective immunity in FVB/N mice against the adoptive transfer of Tg1-1 cells, a neu-expressing tumor cell line generated from a mouse mammary tumor that spontaneously arose in a FVB/N neu-transgenic mouse. The i.m. injection of pNeu_TM or pNeu_E, and to a lesser extent pNeu_N, induced protective immunity against a subsequent challenge with Tg1-1 cells in FVB/N mice. In addition, the coinjection of a plasmid encoding interleukin-2 (designated pIL-2) augmented the efficacy of each of the pNeu plasmids for inducing protective immunity. The plasmid pNeu_TM seemed to be the most effective for inducing anti-neu antibodies. However, the generation of detectable anti-neu antibodies in response to any one of these pNeu plasmids was not enhanced by coinjection of pIL-2 and was not required for protective immunity against Tg1-1 cells. These studies demonstrate that DNA expression vectors encoding soluble or membrane-bound forms of neu lacking the cytoplasmic kinase domain can be effective in inducing protective antitumor immunity.