Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C₁₂-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C₁₂-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C₁₂-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-α secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure−activity profile. From IC₅₀ values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11−13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C₁₂-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-α secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-α driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.