One major concern with the use of antifouling paints is the release of its biocides (mainly copper and zinc) into natural waters, where they may exhibit toxicity to non-target organisms. While many studies have quantified the release of biocides from antifouling paints, very little is known about the physicochemical state of released copper. For proper risk assessment of antifouling paints, characterization of copper released into water is necessary because the physicochemical state determines the metal's environmental fate and effects. In this study, we monitored release of different fractions of copper (dissolved, nano, and bulk) from a commercial copper-based antifouling paint. Release from painted wood and aluminum mini-bars that were submerged in natural waters was monitored for 180 days. Leachates contained both dissolved and particulate copper species. X-ray diffraction and X-ray photoelectron spectroscopy were used to determine the chemical phase of particles in the leachate. The amount of copper released was strongly dependent on water salinity, painted surface, and paint drying time. The presence of nanosized Cu2O particles was confirmed in paint and its leachate using single-particle inductively coupled plasma-mass spectrometry and electron microscopy. Toxicity of paint leachate to a marine phytoplankton was also evaluated.