Two new cobalt corrinoid intermediates, cobalt-precorrin 5A and cobalt-precorrin 5B, have been synthesized with the aid of overexpressed enzymes of the vitamin B-12 pathway of Salmonella enterica serovar typhimurium. These compounds were made in several regioselectively C-13-labeled forms, and their structures have been established by multidimensional NMR spectroscopy. The addition of CbiF to the enzymes known to synthesize cobalt-precorrin 4 resulted in the formation of cobalt-precorrin 5A, and the inclusion of CbiG with CbiF produced cobalt-precorrin 5B, which has allowed us to define the role of these enzymes in the anaerobic biosynthetic pathway. CbiF is the C-11 methylase, and CbiG, an enzyme which shows homology with CobE of the aerobic pathway, is the gene product responsible for the opening of the ring A delta-lactone and extrusion of the "C-2" unit. The discovery of these long-sought intermediates paves the way for defining the final stages of the anaerobic pathway. It is of considerable evolutionary interest that nature uses two distinct pathways to vitamin B-12, both conserved over several billion years and featuring completely different mechanisms for ring-contraction of the porphyrinoid to the corrinoid ring system. Thus the aerobic pathway utilizes molecular oxygen to trigger the events at C-20 leading to contraction and expulsion of the "C-2" unit as acetic acid from a metal-free intermediate, whereas the anaerobic route features internal delivery of oxygen from a carboxylic acid terminus to C-20 followed by extrusion of the "C-2" unit as acetaldehyde, using cobalt complexes as substrates.