The light chain of HLA class I protein (β 2m) has been expressed in Aspergillus nidulans. The cDNA of β 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in β 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of β 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 117 μg/liter of fβ 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human β 2m produced in fungi (fβ 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified fβ 2m from culture supernatants appeared biochemically similar to β 2m obtained from human urine (uβ 2m) as seen in immunoblot analysis. Functionally, fβ 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELIS A for detecting properly folded HLA class I heavy chain and in assays showing cell-surface β 2m exchange into the mouse class I MHC H-2K d. In these experiments the biological activity of fβ 2m was indistinguishable from uβ 2m. The successful expression of biologically active β 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.