The alpha-subunit of the G-protein Gi1 alpha is normally dually acylated at its N-terminus with the saturated fatty acids myristate and palmitate. Inhibition of protein myristoylation by treatment with 2-hydroxymyristate prevented neither the incorporation of [3H]palmitate nor the membrane association of this protein when expressed in the COS cells. Construction of a mutant of Gi1 alpha in which serine-6 was replaced by aspartic acid prevented both myristoylation and palmitoylation, and the expressed protein was found primarily in the cytoplasmic fraction. These data indicate the myristoylation is not an absolute requirement for palmitoylation of Gi1 alpha and that palmitoylation, but not myristoylation, plays a key role in membrane association of this G-protein alpha-subunit.