The activation of murine B cells and macrophages by synthetic lipid A analogues, solubilized with triethylamine and complexed with bovine serum albumin, were investigated in vitro. The analogues used are nonphosphorylated, C-1 or C-4' monophosphorylated and C-1,4' diphosphorylated derivatives of beta-1,6-linked D-glucosamine disaccharide possessing both ester- and amide-bound fatty acid substituents. They were divided into 4 groups, A, B, C and D in terms of the fatty acid substitution. Ester- and amine-bound fatty acids of the analogues are both tetradecanoic acids (C14) in group A, C14 and (R)-3-hydroxytetradecanoic acids (C14-OH) in group B, both C14-OH in group C and C14-OH and (R)-3-tetradecanoyloxytetradecanoic acids in group D. Mitogenic activity was exhibited in spleen cells from C3H/HeN mice by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups B, C and D, but not in cells from C3H/HeJ mice. Nonphosphorylated analogues in groups A, B and C, and C-4' monophosphorylated analogue in group A showed negative mitogenic activity. Only the nonphosphorylated analogue in group D exhibited mitogenic activity in spleen cells from C3H/HeJ mice as well as those from C3H/HeN mice. None of the other analogues exhibited the activity in C3H/HeJ spleen cells. Polyclonal B cell activation activity was exhibited by the C-1 monophosphorylated analogues in groups A and B, and by the C-4' monophosphorylated analogues in groups C and D. Nonphosphorylated analogues in all groups and C-4' monophosphorylated analogues in groups A and B showed negative PBA activity. None of the analogues tested could induce any cytostatic macrophages from thioglycollate-elicited peritoneal macrophages.