Human immunodeficiency virus type 1 (HIV-1) NEF protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or rats. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia coli using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEF gene products failed to induce meiotic maturation when injected into fully grown Xenopus oocytes.