We synthesized and assessed the role of a diacylglycerol (DAG)-containing docosahexaenoic acid (DHA), i.e., 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDHG), in the contraction of guinea pig airway smooth muscle (ASM). We compared its action with 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and 1,2-dioctanoyl-sn-glycerol (1,2-DiC8), a stable DAG analogue. The three DAGs (SAG, SDHG and 1,2-DiC8) induced reversible concentration-dependent contraction of ASM. SDHG induced higher guinea pig ASM contraction than SAG and 1,2-DiC8. The effects of SDHG were blocked, up to different extents, by nifedipine (L-type Ca²⁺ channel blocker). By employing GF-109203X (protein kinase C inhibitor) and lanthanum (La³⁺), a non-selective cation channel blocker, we observed that SDHG evoked ASM contractile response via PKC-dependent and PKC-independent (but Ca²⁺-dependent) pathways. Interestingly, SAG exerted its action only by increasing [Ca²⁺]i and did not require PKC activation. In order to probe the implication of calcium mobilisation, we employed thapsigargin (TG) which also induced ASM contraction in a calcium-dependent manner. SDHG and 1,2-DiC8, in a PKC-dependent manner, induced the phosphorylation of CPI-17 (myosin light chain phosphatase inhibitor of 17 kD). Furthermore, SAG and TG failed to phosphorylate CPI-17 in ASM cells. Our results suggest that different DAG species, produced during a dietary supplementation with fatty acids, could modulate the reactivity of airway smooth muscles in a PKC-dependent and -independent manner, and hence, may play a critical role in health and disease.