Recombinant vaccinia viruses (RVV) designated RVV El, RVV E2, and RVV E3, were constructed to express three different versions of the human T cell leukemia virus type I (HTLV-I) envelope proteins to determine which configuration elicits an optimal antibody response. RVV E1 expressed the native HTLV-I envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while RVV E2 expressed the envelope precursor with the proteolytic cleavage site deleted. The RVV E3 construct expressed only the external surface glycoprotein, gp46. Radioimmunoprecipitation and FACS analysis confirmed that the appropriate envelope proteins were expressed by RVV El-, E2-, and E3-infected cells. Immunization studies were carried out using Balb/c, A/1, and C57BU6 strains of mice. Balb/c mice responded poorly to immunization with all of the three RVV constructs. C57BU6 mice produced neutralizing antibodies in response to immunization with all three constructs, whereas A/J mice developed neutralizing antibodies only when immunized with the RVV E1s construct. The results indicate that the humoral immune responses depend on the form of HTLV-I envelope proteins expressed by each RVV. © 1992.