Although the C3H/HeJ mouse is hyporesponsive to lipopolysaccharides (LPS), certain forms of the lipid A fraction have been shown to stimulate cells from this mouse strain. To determine the role of the oligosaccharide chain length on the lipid A-induced proliferation of C3H/HeJ splenocytes, a panel of glycolipids from R-chemotypes (Re, Rc, and Rd) and a nontoxic monophosphoryl lipid A (MPL) were tested. The MPL cells isolated from the MPL of Salmonella minnesota, Salmonella typhimurium, and the Reglycolipids isolated from Escherichia coli were found to be effective at stimulating the LPS-hyporesponsive spleen cells. A Re-glycolipid isolated from a different strain of E. coli cells was inactive, as where the S. minnesota Rc and Rd chemotypes. Proliferation induced by MPL and the active Re preparations was dose dependent and was inhibited by polymyxin B. Thus, if contamination of the Re-LPS or MPL with lipid A-associated protein occurred, it was below functional levels. The data suggest that the C3H/HeJ spleen cells are capable of responding to certain glycolipids, but they may lack the ability to convert native LPS into a stimulatory signal. In addition, a monosaccharide precursor of lipid A (lipid X), and a monoacyl glucosamine phospholipid derivative of lipid X (MaGP), were capable of inhibiting the proliferation induced by the MPL and Re-glycolipids. These data are compatible with the existence of a spleen cell receptor for lipid A.