An agarose method was used to determine the nature of chemotaxins for bovine leukocytes as related to coliform mastitis. Freshly collected bovine serum or plasma was frequently chemotactic, probably due to spontaneous complement activation. Fresh serum was always rendered chemotactic when activated with zymosan or when heat-killed or viable coliform bacteria were incubated in the serum. The bacteria did not contribute to preexisting activity in the serum. Escherichia coli grown in synthetic media did not generate chemotaxins, and the synthetic peptide formyl-L-methionyl-L-leucyl-L-phenyl-alanine was not chemotactic in the agarose system for bovine leukocytes. Endotoxin-treated serum had variable activity.