The Evi1 transcriptional repressor protein is expressed in a developmentally regulated manner, is essential for normal development, participates in regulating cell proliferation and differentiation of cells of haemopoietic and neuronal origin and contributes to the progression of leukaemia. In this report we describe a new murine Evi1 gene transcript (Delta105) that contains two alternatively spliced regions encoding a 9 amino acid insertion (Rp+9) within the repressor domain (Rp) and a 105 amino acid C-terminal truncation. Abundant levels of the 105 amino acid truncated protein are observed in murine leukaemia cells. The combined primary sequence alterations do not affect the DNA binding, transcriptional repressor or CtBP2 protein binding properties of Evi1 but they do reduce its transforming and cell proliferation stimulating activities. Reduced transforming activity is most likely due to the C-terminal truncation as the activity of Evi1 containing either Rp or Rp+9 is indistinguishable. Both isoforms exist in all murine tissues and cell lines examined. However, only the Rp+9 alternative splice variant is also found in humans and other vertebrates. Murine and human forms of Evi1 with Rp or Rp+9 exist. The additional 9 amino acids are encoded by a conserved 27 nucleotide exon, the overall structural organisation of the gene being preserved in the two species. The function of the Rp+9 and Delta105 splice variants is unknown although the conservation of Rp+9 throughout evolution in vertebrate species suggests it is essential to the broad spectrum of biological activities attributed to this developmentally essential protein.