Regulation of the bovine Saa3 promoter in response to signaling molecules associated with lactation or bacterial infection was assessed using a luciferase reporter system. Although the liver is the primary site for the production of acute phase proteins, typically serum amyloid A1 (SAA1) and serum amyloid A2 (SAA2), analysis of the differential expression of serum amyloid A3 (SAA3) by mammary epithelial cells is limited. Gram-negative bacterial lipopolysaccharide (LPS) and the Gram-positive bacterial lipoteichoic acid (LTA) substantially upregulated transcriptional expression driven by the Saa3 promoter in bovine mammary epithelial cells by 18.5-fold and 12.5-fold, respectively, whereas the lactogenic hormone, prolactin (PRL) stimulated a 3.5-fold increase. The minimal Saa3 promoter fragment that retained responsiveness to LPS, LTA, or PRL was 352 bp in size. A 1056 bp Saa3 promoter region exhibited the highest level of LPS or LTA inducible activity. This activity was 2-fold higher than the constitutive activity obtained with the Simian Virus 40 (SV40) promoter. The 53 bp 5' untranslated region (UTR) in exon 1 of the Saa3 gene enhanced expression levels in response to the stimulants LPS and LTA and the AT-rich region between nt -2571 and -2338 in the Saa3 promoter contained an enhancer that negated a silencer region(s) located between nt -2338 and -1003. Collectively, these data support the proposal that SAA3 serves an important tissue-specific function for the welfare of the mammary gland during both bacterial infection and tissue remodeling.