The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.