High doses of chemotherapeutics in clinical treatment, leading to cell toxicity, can be lowered by co-administration of other immunoregulatory drugs. The aim of this study was to evaluate effects of several derivatives of cyclolinopeptide A (CLA), derived from linen seeds, on the suppressive action of metothrexate (MTX) in a mouse model of humoral immune response in vitro. New CLA analogues 1 and 2, and their linear precursors 3 and 4, containing conformationally restricted dipeptide fragment Phe-Phe or D-Phe-D-Phe with ethylene bridge (-CH(2)-CH(2)-) between phenylalanine nitrogens were synthesized. NMR studies and theoretical calculations showed that introduction of locally constraining fragment into CLA molecule increased its overall conformational flexibility. The bioactivity of new CLA analogues was examined in the mouse model of the in vitro secondary humoral immune response, suppressed by methotrexate (MTX). The results revealed differential actions of the peptides such as 1/augmentation of the suppressive activity of MTX or 2/antagonistic effects of the peptides on MTX-induced suppression. Potential advantages for the application of CLA-derived peptides in therapy and structure-activity relationships were discussed.