Zostera marina (eelgrass) is an important ecological component of many shallow, temperate lagoons. Evidence suggests that Z. marina has a high bicarbonate utilization capability, which could be promoted by possible proton extrusion and the consequent formation of an ‘acid zone’ in the apoplastic space (unstirred layer) of its leaves. It has been found that 50 mM of the buffer Tris significantly inhibited the photosynthetic O2 evolution of Z. marina and it was proposed that this was because of Tris's ability to bond with protons outside the cell wall. To investigate if H+ played an important role in the photosynthetic carbon utilization of Z. marina, it is very important to simultaneously monitor the photosynthesis status and possible H+ fluxes. However, probably because of the lack of suitable techniques, this has never been attempted. In this study, experiments were undertaken on Z. marina by monitoring H+ and O2 fluxes and the relative electron transport rates during light–dark transition. During stable photosynthesis, in addition to an obvious O2 outflow, there was a significant net H+ influx connected to Z. marina photosynthesis. The inhibitory effects of both Tris and respiration inhibitors on apparent O2 evolution of Z. marina were confirmed. However, evidence did not support the proposed Tris inhibition mechanism.