Diacylglycerol kinases are key modulators of levels of diacylglycerol, a second messenger involved in a variety of cellular responses to extracellular stimuli. A number of diacylglycerol kinases encoded by separate genes are present in mammalian genomes. We have cloned cDNAs encoding several isoforms of the human homologue of the rat diacylglycerol kinase beta gene and characterized two such isoforms that differ at their carboxyl terminus through alternative splicing and the usage of different polyadenylation signals. Quantitative analysis of gene expression in a panel of human tissue cDNAs revealed that transcripts corresponding to both isoforms are co-expressed in central nervous system tissues and in the uterus, with one variant being expressed at relatively higher levels. As green fluorescent protein fusions, the two isoforms displayed localization to different subcellular compartments, with one variant being associated with the plasma membrane, while the other isoform was predominantly localized within the cytoplasm. Differences were also observed in their subcellular localization in response to phorbol ester stimulation. Enzymatic assays demonstrated that the two isoforms display comparable diacylglycerol kinase activities. Therefore, the human diacylglycerol kinase beta gene can generate several enzyme isoforms, which can display different expression levels and subcellular localization but similar enzymatic activities in vitro.