The molecular cloning and initial characterization of the complementary DNA encoding the chicken homologue of the human retinoblastoma susceptibility gene product Rb are described. Chicken Rb (chRb) was found to be highly homologous to human, mouse, and Xenopus Rb in primary amino acid sequence, underlining its conserved function in cell cycle control in higher eukaryotes. The highest level of homology was found within the Rb "pocket domains" and at the Rb COOH terminus, whereas the extreme NH2 terminus of chRb is divergent. We also show that transcription of chRb initiates at multiple start sites within a DNA segment which represents a CpG island. ChRb is transcribed as a 4.7-kilobase (kb) mRNA species in a variety of normal chicken tissues and oncogene-transformed hematopoietic cells. However, in v-rel-transformed cell lines, a variant 3.1-kb Rb-specific mRNA was detected in addition to the 4.7-kb transcript. ChRb is expressed as a 104 kilodalton protein and is therefore slightly smaller than the mammalian Rb proteins.