The envelopes of two highly divergent oncoviruses, human T-cell leukemia virus type 1 (HTLV-1) and Friend murine leukemia virus (F-MuLV), have distinct patterns of cellular receptor recognition, fusion, and syncytium formation. To analyze the influence of the transmembrane envelope subunit (TM) on fusogenic properties, we substituted either the entire TM or distinct domains from F-MuLV for the corresponding domains in the HTLV-1 envelope. Parental, chimeric, and truncated envelopes cloned into a eukaryotic expression vector were monitored for fusogenic potential in human, rat, and murine indicator cell lines by using a quantitative assay. This highly sensitive assay allowed us to assess the fusogenic properties and syncytium-forming abilities of the HTLV-1 envelope in murine NIH 3T3 cells. All chimeric envelopes containing extracellular sequences of the F-MuLV TM were blocked in their maturation process. Although deletions of the HTLV-1 cytoplasmic domain, alone and in combination with the membrane-spanning domain, did not prevent envelope cell surface expression, they impaired and suppressed fusogenic properties, respectively. In contrast, envelopes carrying substitutions of membrane-spanning and cytoplasmic domains were highly fusogenic. Our results indicate that these two domains in F-MuLV and HTLV-1 constitute structural entities with similar fusogenic properties. However, in the absence of a cytoplasmic domain, the F-MuLV membrane-spanning domain appeared to confer weaker fusogenic properties than the HTLV-1 membrane-spanning domain.