Bovine apo-lactoferrin (apo-Lf) was added to in vitro cultures of eight strains of coliform bacteria associated with bovine mastitis. As little as 0.02 mg of APO-Lf per ml resulted in marked inhibition of growth of all coliforms. Growth inhibition was lost if saturated Lf or iron plus apo-Lf was added to the synthetic medium. The inhibition of growth increased as the concentration of apo-Lf increased from 0.02 to 0.2 mg/ml for Klebsiella pneumoniae (OARDC-A1), Klebsiella spp. (K1-21), and Aerobacter aerogenes (55-12222) and 2 mg/ml for A. aerogenes (76-2414-1), Escherichia coli (60-Lilly), E. coli (66-S16), and Klebsiella spp. (K6-24). As the concentration of apo-Lf was increased above 0.2 or 2 mg/ml, there was less inhibition of growth except for E. coli (33-C4). Apo-Lf at 20 mg/ml was bactericidal for E. coli (33-C4). Results are compatible with the hypothesis that coliform bacteria respond to low-iron environments by production of iron-sequestering agents that complete effectively with apo-Lf for free iron. Addition of apo-Lf plus citrate resulted in loss of growth inhibition. The molar ratio (citrate to apo-Lf) was found to be more important than the absolute concentration of either component. A ratio of 75 resulted in 50% growth inhibition, whereas ratios of 300 and greater resulted in less than 10% growth inhibition. These results suggest that the ratio of citrate to Lf would be important in evaluating Lf as a nonspecific protective factor of bovine mammary secretions.