Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO 2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO 2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonylCoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA ؉ NADPH ؉ H ؉ 3 malonate-semialdehyde ؉ CoA ؉ NADP ؉ . In its native state it is associated with small RNA. Its activity was stimulated by Mg 2؉and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (K m ‫؍‬ 25 M) and malonyl-CoA (K m ‫؍‬ 40 M). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonylCoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.