Novel phospholipid/polydiacetylene vesicles are reported herein, and their optimized sensing performance and the effects of lactate counterions and pH are elucidated. Vesicles comprising 10,12-pentacosadyinoic acid (PCDA), PCDA/cholesterol/ sphingomyelin (PCDA/CHO/SPH), PCDA/1,2-dimyristoyl-sn-glycero-3-phosphocholine (PCDA/DMPC), and PCDA/1,2dimyristoyl-sn-glycero-3-phosphoglycerol (PCDA/DMPG) were synthesized, and their diameters and colorimetric responses induced by sodium and calcium lactates were assessed. The vesicles were characterized at as-synthesized pH as well as at reduced (pH < pK a ) and increased (pH > pK a ) proton activities. This contribution demonstrates that the presence of lactate caused the colorimetric transition in PDA vesicles and that the interaction between these components depended upon vesicle composition, medium pH, and lactate counterion. Variations in vesicle diameter arising from calcium or sodium lactate concentrations are probably related to the disruption of vesicle surfaces, which does not change the internal molecular packing. Therefore, vesicle growth was not always responsible for the colorimetric transitions, which in fact were assigned to conformational changes of the PDA molecules.