The enzymic conversion of dolichol into dolichoic acid has been studied in bovine thyroid subcellular fractions using [1-3H]dolichol as a substrate. The presence of conversion activity could be demonstrated in both the mitochondrial- and supernatant fractions. Investigation of cofactor requirements revealed that NAD+ was essential for reaching optimal activity. From kinetic studies Km-values of 3.5-4 microM and 0.29 mM could be calculated for, respectively, dolichol and NAD+ using the mitochondrial fraction as an enzyme source. No inhibitory effects from ethanol or pyrazole were detected suggesting that alcohol dehydrogenase is not involved in the dolichol-->dolichoate conversion as observed in a bovine thyroid mitochondrial fraction. From inhibitor studies the conversion system behaves distinctly differently from the NADP(+)-depending microsomal oxidoreductase as well as from catalase. The conversion activity in the supernatant on the other hand must be ascribed, at least partially, to a side activity of alcohol dehydrogenase.