Several factors, including the exposure of the negatively charged PL and transmembrane potential (TMP), may affect the binding of merocyanine 540 dye (MC540) to membrane lipids. Our aim was to quantify the significance of each of these two determinants in MC540 interactions with phosphatidylserine:phosphatidylcholine (PS/PC) vesicles. The effects of the altered PS content (PS/PC molar ratio: 5:95, 10:90 and 20:80) and TMP on MC540 binding were monitored using flow cytometry. Rapid [K(+)] flux across the vesicle membrane lipid bilayer was generated using valinomycin. We showed that the increased PS content leads to attenuated MC540 binding, while having no influence on the dynamic parameters of PS/PC vesicle membranes (electron spin resonance (ESR) spectrometry). Higher [K(+)](out) makes PS/PC liposomes bind more MC540, which implies that TMP-which becomes more positive inside the vesicles-favours the interactions of MC540 with the PL bilayer. Overall, the variability attributed to MC540-PL interactions is explained only to a minor extent by the generated TMP (7%) and largely by the variations in PS content (by up to 60%). In conclusion, the content of negatively charged PL is more important than TMP in determining the interactions of MC540 with PS/PC membranes.