The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.