Near-IR FT-Raman spectra of B-12 imidazole derivatives and cobalamins in aqueous solution were compared since there is now strong evidence that, in human Bit-dependent enzymes, the 5,6-dimethylbenzimidazole (DMBz) is replaced by imidazole from a histidine in the protein. Derivatives studied include methylcobalamin [MeCbl (DMBz base-on) and MeCbl(+) (base-off by acidification to protonate the DMBz)], methylaquacobinamide (MeCbi(+)) [Cbi’s have the DMBz-bearing nucleotide loop removed by hydrolysis], and Me(N-acetylhistidine)Cbi [coordinated through imidazole in Me(N-AcHis)Cbi at pH 10 and imidazolate in Me(N-AcHis)Cbi(-) in 1 M NaOH]. Several marker bands changed with changes in the axial ligand trans to the methyl group, The frequency of the Co-CH3 stretching mode at similar to 505 cm(-1) (assigned by isotopic shift using -CD3) was similar for all MeCbl and MeCbi species; thus, the trans ligand, including the very powerful electron-donating imidazolate species, has little effect on Co-C bond strength. In contrast, the peak height of the Co-CH3 band, relative to the corrin long-axis mode band at 1495 cm(-1), consistently increased 2-fold upon coordination of an N-donor ligand to MeCbi(+) and MeCbl(+).