Adenylate kinases (AKs) are ubiquitous phosphotransferases that contribute to homeostasis of adenine nucleotide composition in cells. Six AK isoforms were found in vertebrates. We report that soluble AK isoform 1 is expressed in the cytosol of bovine retina consistently devoid of rod outer segments. Immunoblotting analysis with a polyclonal antibody raised against soluble adenylate kinase and subsequent sequencing of eluted peptide by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry allowed enzyme isolation by joining purification methods to two-dimensional electrophoresis. In this study, we found that cytosolic adenylate kinase isoform 1 is expressed in bovine retina. Cytoplasmic AK1 would physiologically contribute to retinal energy metabolism.