Estimates of copepod secondary production are of great importance to infer the global organic matter fluxes in aquatic ecosystems and species-specific responses of zooplankton to hydrologic variability. However, there is still no routine method to determine copepods secondary production in order to eliminate time consuming experimental analyses. Therefore, we determined whether there is a correlation between Egg Production Rates (EPR) and RNA:DNA ratios of Acartia, by measuring their seasonal and spatial variability and the influence of environmental factors for Acartia sp. collected in the the Guadiana river estuary. EPR of A. tonsa was positively related with chlorophyll a concentration, freshwater inflow and biomass of dinoflagellates, while A. clausi was only related to dinoflagellates. Dinoflagellates seem to be the optimal food item influencing the reproduction of both Acartia species in the studied area. The biochemical index RNA:DNA was positively related to EPR, indicating that it is a good proxy of copepod production and a promising method to use in the future to estimate secondary production.