Mammalian l-isomer dehydrogenases represent an expansive and well characterized class of metabolic enzymes. Surprisingly, little is known regarding their evolutionarily distinct counterparts, d-isomer dehydrogenases, since few mammalian d-isomer 2-hydroxy acid enzymes have been isolated. Here we present the identification and initial characterization of putative human and murine d-lactate dehydrogenases (DLD) that can interact with the muscle-specific cysteine-rich protein CRP3/MLP. Sequence analysis reveals that the human and mouse transcripts encode novel proteins that display strong similarities to the yeast d-lactate dehydrogenase proteins DLD1, AIP2, and YEL071W. Expression analysis of the mammalian proteins indicates widespread distribution with transcripts present in striated muscle tissues and a variety of other tissue types. Immunofluorescence subcellular localization of the mouse DLD protein indicates that it resides within mitochondria, a feature shared by many dehydrogenases. The identification of the human and mouse DLD clones provides new insight regarding the activity of d-isomer-specific enzymes in mammalian cells.