A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A(2) (sPLA(2)). To investigate the unexpected sPLA(2) activity, we have compared three different phospholipids by using fluorescence techniques and HPLC, namely: (R)-1,2-dipalmitoyl-glycero-3-phosphocholine (hereafter referred to as 1R), (R)-1-O-hexadecyl-2-palmitoyl-glycero-3-phoshocholine (2R), and (S)-1-O-hexadecyl-3-palmitoyl-glycero-2-phosphocholine (3S). Furthermore, to understand the underlying mechanisms for the observed differences, we have performed molecular dynamics simulations to clarify on a structural level the substrate specificity of sPLA(2) toward phospholipid analogues with their head groups in the 2-position of the glycerol backbone. We have studied the lipids above 1R, 2R, and 3S as well as their enantiomers 1S, 2S, and 3R. In the simulations of sPLA(2)-1S and sPLA(2)-3R, structural distortion in the binding cleft induced by the phospholipids showed that these are not substrates for sPLA(2). In the case of the phospholipids 1R, 2R, and 3S, our simulations revealed that the difference observed experimentally in sPLA(2) activity might be caused by reduced access of water molecules to the active site. We have monitored the number of water molecules that enter the active site region for the different sPLA(2)-phospholipid complexes and found that the probability of a water molecule reaching the correct position such that hydrolysis can occur is reduced for the unnatural lipids. The relative water count follows 1R > 2R > 3S. This is in good agreement with experimental data that indicate the same trend for sPLA(2) activity: 1R > 2R > 3S.