Aims: The objective of this study was to determine the combined effects of water activity (a(w)), chemical treatment and temperature on Salmonella and Escherichia coli O157:H7 inoculated onto alfalfa seeds. Methods and Results: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 and adjusted to various a(w) values were subjected to simultaneous and separate treatments with chemicals and heat. The rate of death of both pathogens was correlated with increased a(w) (0.15-0.60) and temperature (5-37degreesC) over a 52-week storage period. Higher seed a(w) enhanced the inactivation of pathogens on seeds heated at 50-70degreesC for up to 24 h. Treatment of seeds with water, 1% Ca(OH)(2), 1% Tween 80, 1% Ca(OH)(2) Plus 1% Tween 80 or 40 mg l(-1) Tsunami 200(R) at 23 or 55degreesC for 2 min significantly (alpha = 0.05) reduced populations of Salmonella and E. coli O157:H7. Conclusions: Overall, at the combinations of temperature and concentrations of chemicals tested, 1% Ca(OH)(2) was most effective in killing Salmonella and E. coli O157:H7 without reducing seed viability. Significance and Impact of the Study: None of the treatments evaluated in this study, whether applied separately or in combination, eliminated Salmonella or E. coli O157:H7 on alfalfa seeds without sacrificing the viability of the seeds. It remains essential that practices to prevent the contamination of alfalfa seeds be strictly followed in order to minimize the risk of Salmonella and E. coli O157:H7 infections associated with sprouts produced from these seeds.