K99, a presumed colonizing factor of enterotoxigenic E. coli of calf origin, has been purified. K99 was removed from K99+ bacteria by salt extraction and subsequently purified by ammonium sulfate precipitation and column chromatography on diethylaminoethyl Sephadex. The purified material was homogenous in size, having an s(20,w) of 13 to 15 S. It was composed of two subunits: a major component with a molecular weight of 22,500 and a minor component of 29,500. When observed in the electron microscope, K99 appeared to be rod shaped, with a strong tendency for self aggregation. At concentrations where aggregation was minimized, individual rods were observed with diameters of 8.4 nm and mean lengths of 130 nm. Based on the subunit structure, exterior location, and rod like shape of K99, it is concluded that K99 is a pilus or piluslike structure. Chemically, K99 is composed primarily of protein and has an isoelectric point of greater than 10. Purified K99 did not hemagglutinate guinea pig erythrocytes.