cDNAs encoding the precursor of the alpha-subunit of human electron transfer flavoprotein (p alpha-ETF) were cloned and sequenced. The cDNAs span 1,300 base pairs and include the entire coding region of 333 amino acids. The identity of the p alpha-ETF clones was confirmed by hybrid selected translation, by transcription/translation of a cDNA, and by mitochondrial processing of the protein produced by these translations. The identity of the cDNA clones was further confirmed by matching the amino acid sequence deduced from the nucleotide sequence of the cDNAs to amino acid sequences determined from seven tryptic peptides prepared from purified rat alpha-subunit of electron transfer flavoprotein (alpha-ETF). Ninety-eight of 105 amino acids from these rat alpha-ETF tryptic peptides matched with those deduced from the human cDNAs. The seven amino acid substitutions are presumably due to species difference. The calculated molecular weight of the human alpha-ETF precursor was 35,084. The amino-terminal amino acid of the mature protein could not be determined by amino-terminal sequencing, presumably due to blockage and, therefore, an accurate molecular weight of mature alpha-ETF could not be calculated. The molecular sizes of the precursor and mature alpha-ETF have been estimated to be 35 and 32 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.