Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.