We previously found a new endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. We have cloned and sequenced a cDNA (2989 bp) encoding the ASTP protein. An open reading frame of 524 amino acids encodes a protein with a molecular weight of 57 kDa. Amino-acid sequences of six peptides obtained by Edman degradation of various cleavage products of the purified ASTP were identified in the cDNA-derived sequence. Northern analysis of mRNA from rat liver revealed a major band of about 3.8 kb. Database searches show no homology to other known proteins from mammalian origin, however, the amino-acid sequence in the middle portion of ASTP protein shows 48% homology to glycerol kinase from Escherichia coli. The region of homology to other species may help to define sites of protein-protein interaction, as well as sites of possible interaction of ASTP with ATP.