Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pHi) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pHi range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pHe), as well as between the pHi and the pH of the diluent. The pHi change post-activation in seawater (ASW) depended on the initial pHe of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pHe < 8.0; an acidification when pHe > 8, and no pHi variation when pHe was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pHi changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation.