Rat liver isoleucyl-tRNA formation in the presence of more than 0.6mM Mg²⁺ was inhibited by smooth and rough endoplasmic reticula. The inhibition was partially prevented by ATP, spermine, oligomycin, and Triton X-100. This suggests that microsomal ATPases are responsible for the inhibition of isoleucyl-tRNA formation. Among the microsomal ATPases, an oligomycin-sensitive ATPase was found to be inhibited by spermine. The inhibition of the oligomycin-sensitive ATPase by spermine explains well its partial prevention of the inhibition by endoplasmic reticulum of isoleucyl-tRNA formation.