The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M(r) 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.