The envelope protein of the human T-cell leukemia virus type I (HTLV-I) is highly conserved among the isolates sequenced so far, as opposed to what is observed for the human immunodeficiency virus (HIV) envelope. By linker insertion scanning, we have produced 33 random mutations along the HTLV-I envelope gene, cloned into a eukaryotic expression vector. The resulting envelope products were analysed by immunoprecipitation and syncytia formation after transfection into COS-1 cells. We show here that 25 out of 33 mutations result in a non-functional envelope product as assessed by the lack of ability to form syncytia. In the majority of these mutants, the processing of the envelope gp61 precursor into the mature gp45 and gp20 proteins was affected. We propose that conformational constraints for processing and fusion abilities tend to limit the variability of the HTLV-I envelope. In three mutants, processing was observed but no syncytia were formed. These mutations might affect regions important for HTLV-I envelope functions, such as the receptor binding region.