Human spuma retrovirus (HSRV) belongs to retroviruses that possess a complex genome organization. HSRV carries at least three extra genes in the region between env and the 3' long terminal repeat, which are not found in simple retroviruses. Via alternative splicing, these HSRV genes can encode several proteins. To genetically study the requirements of these viral proteins for viral replication in tissue cultures, a number of mutant viruses were constructed from an infectious molecular clone of HSRV. All mutants grew normally in the cell lines tested, except for those lacking transcriptional transactivator activity. By reporter-based transient assay systems, no Rev/Rex equivalent was detected in the HSRV proteins.