We report the cloning of a novel murine cDNA, LEK1, that is related to human CENP-F and mitosin and more distantly to chicken CMF1. The proteins from these three organisms have significant homology, yet differ in their temporal, spatial, and subcellular localizations. The human proteins bind the kinetochore in mitotic cells, whereas the chicken protein is found only in skeletal and cardiac muscle and is developmentally regulated. Mouse LEK1 is a single copy gene that codes for two developmentally regulated transcripts. The LEK1 protein is expressed early and ubiquitously in mouse development and is generally down-regulated as development proceeds in a manner that correlates to a cessation of mitosis. In adult tissues, the LEK1 protein is detected exclusively in the pronucleus of the oocyte and was not observed in other actively dividing tissues. Subcellular localization revealed that the LEK1 protein in mitotic cells does not bind the kinetochore. From these data, we hypothesize that chicken CMF1, human CENP-F, mitosin, and mouse LEK1 are members of an emerging family of genes that have important and functionally distinct roles in development and cell division.