1. Platelet-activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH((i)), specifically if these changes in pH are the result of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway activation in bovine neutrophils. 2. PAF caused intracellular alkalinization in 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester-loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride-sensitive Na(+)/H(+) exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). 3. PAF 100 nm induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. 4. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular-signal-regulated protein kinase) with an EC(50) of 30 and 13 nm, respectively. 5. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. 6. Our results suggest that PAF induces intracellular alkalinization via PI3K-MAPK activation. This effect is upstream regulated by PAF receptor, PTX-sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.