The effects of the insecticide chlorpyrifos were studied in plankton-dominated mesocosms under Mediterranean conditions. Chlorpyrifos was applied four times at 1 week intervals at nominal concentrations of 0.033, 0.1, 0.33, and 1 microg/l simulating repeated agricultural applications. The lowest 7 days time weighted averaged concentrations (TWAC) during the 28 days exposure period were calculated using the FOCUS equation to express the no observed effect concentration (NOEC) values. At population level the lowest NOEC calculated was 0.012 microg/l (treatment concentration 0.033 microg/l). The most affected taxon was Cladocera (Daphnia group galeata) followed by Copepoda (cyclopoids and nauplii). No effects were observed on phytoplankton (chlorophyll-a biomass) at any treatment level. The smallest NOEC(community) calculated by means of multivariate techniques was 0.1 microg/l when expressed in terms of the nominal treatment level and 0.074 microg/l when based on the lowest 7 days TWA concentration during the 28 days application period. Indirect effects on zooplankton populations were observed due to shifts in competition and predation between populations. Compared with previous micro/mesocosm experiments simulating a single application exposure regime, results from our study revealed a lower threshold level for the most sensitive measurement endpoint (difference a factor of three (in terms of nominal treatment level), more severe indirect effects and longer recovery periods of the affected populations (> 13 weeks in the test systems treated with 1 microg/l). These differences could be attributed to the repeated pulse exposure scenario approach designed for our studies together with the particular climatic conditions involving our Mediterranean mesocosms (i.e., temperature, cladocerans life history, and algae blooms).