In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca(2+)-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La(3+)-dependent. Despite the absence of Ca(2+) in the medium strain AM1 was able to grow on methanol in the presence of La(3+). Addition of La(3+) increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La(3+), and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La(3+) as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca(2+), but was able to grow after supplementation with La(3+). Taken together, these results show that XoxF1 participates in methanol metabolism as a La(3+)-dependent MDH in strain AM1.