The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB.