Aims: To determine the effectiveness of combined treatments with chemicals, heat and ultrasound in killing or removing Salmonella and Escherichia coli O157:H7 on alfalfa seeds intended for sprout production. Methods and Results: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 were treated with ultrasound (38.5-40.5 kHz) in solutions containing 1% Ca(OH)(2), 1% Tween 80, 1% Ca(OH)(2) Plus 1% Tween 80, 160 mug ml(-1) Tsunami(R) 200 and 0.5% Fit(R) at 23 and 55degreesC for 2 and 5 min. Highest reductions were in chemical solutions at 55degreesC, but seed viability was also reduced compared with treatment at 23degreesC. Inactivation of Salmonella and E. coli O157:H7 was generally enhanced by simultaneous treatments with ultrasound, chemicals and heat. Conclusions: Ultrasound treatment, in combination with chemicals and heat, had a modest enhancing effect on the effectiveness of chemicals in killing or removing pathogens on alfalfa seeds. Overall, treatment with 1% Ca(OH)(2) was most effective in killing Salmonella and E. coli O157:H7. Significance and Impact of the Study: The use of 1% Ca(OH)(2) instead of 20 000 mug ml(-1) chlorine, which is currently recommended as a sanitizer for seeds intended for sprout production in the US, should be considered. Ultrasound treatment of alfalfa seeds containing Salmonella or E. coli O157:H7, in combination with chemical treatment, contributes to achieving greater reductions in populations of these pathogens, thereby reducing the risk of contamination and the presence of pathogens in sprouts produced from these seeds.