We synthesized seven new analogues of KRN7000: RCAI-133 and 154-159. RCAI-154, 156 and 158 are secondary-amide analogues having a hydroxy, a methyl or two methyl groups at the alpha-position of a linear C-18-acyl chain, respectively. RCAI-155, 157 and 159 are corresponding N-methylated tertiary amide analogues, and RCAI-133 is the N-methylated KRN7000. Among them, a PBS solution of RCAI-133 induced mouse lymphocytes to produce Th2-biasing cytokines in vivo, while RCAI-154-159 showed only weak or almost no immunostimulatory activity. Therefore, N-methylation led the glycolipid to produce predominantly Th2-type cytokines, while acyl alpha-substitution was detrimental to activity.