Conclusion Much progress has been made since the identification of Nef (or 3′ orf as it was then known) as a protein product of HIV-1 in 1985 (Allan et al., 1985). As I have discussed in this review, a decade of studies has shown that Nef has a number of clearly defined in vitro biological functions and a critical role in virus pathogenesis in vivo. There are several tasks to be undertaken in the foreseeable future. Firstly, the biochemical mechanisms of Nef function in vitro must be accurately defined, hopefully leading to the development of chemotherapeutic antiviral agents. Implicit in this analysis must be the investigation of the influence of Nef on virus replication in cell types other than T cells that are known to be infected by HIV, e.g. macrophages and glial cells. It is conceivable that the biochemistry of Nef function in these cell types may be distinct from that in T cells. With regard to macrophages it is pertinent that Nef apparently interacts with a macrophage-specific tyrosine kinase, Hck, with high affinity (Saksela et al., 1995). Secondly, the relative contribution of each of the in vitro functions to the role of Nef in vivo must be identified. Intuitively, it is to be expected that both CD4 down-modulation and the enhancement of virus infectivity will be important for pathogenesis of HIV infection. However, formal proof of this must be obtained. It will also be important to ascertain whether these in vitro functions are truly independent. Although both CD4 down-modulation and enhancement of virus infectivity can occur independently under in vitro conditions, it cannot be ruled out that they are linked in some way in vivo. They may be manifestations of an underlying biochemical mechanism, as yet unidentified. The most obvious grey area concerning Nef function is the effect of Nef on signal transduction pathways. This aspect of Nef embraces the most controversy and disagreement and clear interpretation of the available data is impossible. Above all, the priority here must be to demonstrate that any effects of Nef observed in vitro are relevant to virus replication and not merely artifacts of expression of a heterologous protein. That is the real challenge for the future.