Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.