Using a subtractive hybridization procedure, we isolated a cDNA clone encoding elongation factor 1 alpha (EF-1 alpha) from chicken cells devoid of mitochondrial (mt) DNA (rho0). The sequence encodes 1691 nucleotide (nt) residues and contains an open reading frame of 463 codons. Compared with the sequences from human, mouse and Xenopus laevis, the highest degree of sequence identity is detected in the 3' untranslated (> 90%) and coding (> 85%) regions. The gene evolved mainly by transitions occurring at the third codon position. Most transitions are silent and amino acid (aa) sequence identities are greater than 95%. Comparison of the protein domains interacting with cellular components (GTP/GDP, tRNAs and beta-actin) reveals that they are highly conserved in species belonging to the four traditional eukaryotic kingdoms. The expression of the EF-1 alpha transcript is elevated in chicken rho0 cells. A single RNA band at 1800 nt is observed in both parental and rho0 cells. Southern blot analysis of restricted DNA from chick embryo fibroblasts (CEF) suggests that only one gene encoding EF-1 alpha exists in the chicken genome.