Transgenic Bt maize plants are genetically modified to contain genes of Bacillus thuringiensis that encode for δ-endotoxins (Cry1Ab protein) that have insecticidal properties. These endotoxins target certain lepidopteran pests of maize. There are several entry routes by which Cry proteins enter the aquatic ecosystem in which aquatic organisms are exposed to these proteins. The main route is through plant debris such as leaves, stalks and postharvest detritus that are transported by means of run-off, rain and wind. While several studies have been conducted on the fate of Cry1Ab protein in terrestrial ecosystems, little is known of the release rates of Cry1Ab proteins from maize plant tissues that end up in aquatic ecosystems. In this study, leaves of Bt-maize and its isoline were submerged in containers filled with deionised or borehole water for a period of 16 days, and kept at 3 different temperatures (10±1, 21±1 and 30±1°C). Samples were collected at 1, 2, 4, 8, 16, 24, 48, 96, 192 and 384 h post submersion and analysed for Cry protein content using enzyme-linked immunosorbent assay (ELISA). The release of Cry protein from submerged maize leaves was influenced by temperature, and duration of immersion. An increase in Cry protein levels in the water was observed from the first hour onwards in both water types until the end of the experiment. The highest concentration of Cry protein was found at 30°C. This study showed that temperature and time period influence the release rate of Cry proteins from dried leaf matter into the aquatic environment.