The objective of this study was to evaluate the effect of smooth (S) and rough (R) forms of lipopolysaccharide (LPS) on gene expression in bovine blood neutrophils. Isolated neutrophils (10(7) cells/mL) were treated with Escherichia coli LPS serotype O111:B4 (S+R) and R forms (Ra, Rd, or Rc). Flow cytometry was used to assess surface expression of toll-like receptor 4 (TLR-4). Specific primers for IL-8, tumor necrosis factor-alpha (TNF-alpha), IL-1beta, cluster of differentiation antigen 14 (CD14), and natural resistance associated macrophage protein 1 (Nramp1) were used to assess transcription. Secretion of IL-8, TNF-alpha, or IL-1beta was determined using ELISA kits. Both S and R forms of LPS bound to neutrophils. Exposure induced increased surface expression of TLR-4. No TLR-4 or CD14 mRNA was detected but transcripts for IL-8, TNF-alpha, IL-1beta, and Nramp1 were detected. Secretion of IL-8 and TNF-alpha but not IL-1beta was observed following treatment with R forms of LPS. The longest R form tested (Ra) increased RNA purity and IL-8 and TNF-alpha secretion in bovine neutrophils. The Rd form increased TLR-4 expression and reduced IL-8 and TNF-alpha secretion. Exposure to LPS induced increased cell surface expression of TLR-4 and enhanced expression of IL-8 genes. Enhanced TLR-4 activity by LPS was not dependent on transcriptional activation. Recruitment of TLR-4 to the cell membrane may account for increased cell surface expression. A CD14-independent, TLR-4-dependent pathway may be important in the response to different forms of LPS by bovine neutrophils.