The primary structure of the 49 K subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The sequence lengths correlate to a molecular mass of 54,002 daltons for the preprotein and 49,239 daltons for the mature protein. The presequence consists of 42 amino acids of typical composition for sequences which target nuclear-encoded proteins into mitochondria. The mature protein consists of 436 amino acids and shows 64% similarity to a 49 K subunit of bovine heart NADH:ubiquinone reductase and 33% to a predicted translation product of an open reading frame in the chloroplast DNAs of Marchantia polymorpha and Nicotiana tabacum. Evidence for an iron-sulfur cluster in the subunit is discussed.