Characterization of surface-active organic substances has been performed in the North Adriatic Sea, in surface and bottom layers, during the year 2000. Surfaceactive substances (SAS), their hydrophobicity and acidity, were estimated with alternating current voltammetry (in-phase mode) using o-nitrophenol as an electrochemical probe. Depending on hydrophobicity and acidity of present SAS electrochemical characteristics of the o-nitrophenol probe are changing in a specific way. Normalization of the change of prepeak height as an indication of organic matter acidity to shift of peak potential as a measure of hydrophobicity give indication of relative acidity of present SAS. Experiment with model substances showed that humic and fulvic acids contribute the highest to the relative acidity, while biogenic surface-active organic substances as proteins, polysaccharides and lipids contribute lower to the relative acidity.Surface water layer is characterized with higher phytoplankton excretion during whole year concluded from higher contribution of biogenic organic substances to surfactant activity. Humic and fulvic substances dominate in surfactant activity in the bottom layer. Proteinaceous material is found in a heterodispersed fraction during supposed phytoplankton blooms that were in March, July, September and even December, in the Northern Adriatic, connected to unexpectedly high temperatures. Surfactant activity of particulate fraction was found to be mainly less than 10 %. Exceptions were obtained in April and October when surfactant activity of particulate fraction contributed up to 55 and 20 %, respectively. 2