A patient has been described with methylmalonic aciduria because of an inability to release free vitamin B12 from lysosomes. Complementation analysis was performed using fibroblasts from this patient and those from patients having previously described mutations causing methylmalonic aciduria (mut, cblA, cblB, cblC, and cblD). Incorporation of label from [1-14C]propionate into acid-precipitable material was elevated in heterokaryons formed by polyethylene glycol (PEG) treatment of mixed cultures of cells from the patient and all other complementation groups as compared to the incorporation in parallel cultures not treated with PEG. These results indicate that complementation occurred in all cases and support the assignment of the patient to a new complementation group that has been designated cblF.