Canarypox viruses undergo abortive replication in mammalian cells. Despite this restriction on replication in mammalian cells, significant immune responses have been shown in animals and in humans receiving recombinant canarypox vaccine vectors expressing heterologous immunogens. A recombinant canarypox vaccine candidate (vCP205), which expresses human immunodeficiency virus (HIV)-1 Gag, Env, and protease proteins, is presently under investigation in phase I and phase II human trials in the United States and elsewhere. In this study, the ability of vCP205 to elicit HIV Gag-Env pseudovirion formation in avian and mammalian cells was investigated. Gag-Env pseudovirions were produced from both avian and mammalian cell lines infected by this vaccine vector. A subset of mammalian cells was identified in which pseudovirion production and release was very efficient, surpassing the production from infected avian cells. The production of Gag-Env pseudovirions by canarypox HIV vaccine vectors may have important implications for future HIV vaccine design.