The objective of this study was to investigate the effect of uptake of different commonly consumed long chain fatty acids on superoxide (O(2)(-)), nitric oxide (NO) production, and ability to kill Salmonella enterica serotype typhimurium (S. typhimurium) by chicken macrophages (HD11 cells). All the fatty acids were taken up by HD11 cells with stearic acid uptake higher than polyunsaturated fatty acids. Uptake of green fluorescent protein-labeled bacteria and the viability of HD11 cells (measured by flow cytometry) was not affected by any of the fatty acids tested. Bacterial clearance (measured by the plating of sorted viable infected cells) was significantly higher with n-3 fatty acids alpha-linolenic acid (ALA) and docosahexanoic acid (DHA). However, stearic acid (SA) and the n-6 fatty acid, arachidonic acid (ARA) did not influence S. typhimurium killing by HD11 cells. The improved S. typhimurium clearance by ALA and DHA was not associated with increased NO or O(2)(-) production by HD11 cells. These results suggest a role for n-3 polyunsaturated fatty acids in Salmonella clearance by chicken macrophages however in vivo studies are essential to confirm their efficacy in controlling Salmonella infection in chickens and contamination in shell eggs.