Although it is known for many years, that transformation products (TPs) of pesticides are often more persistent, mobile, and sometimes more toxic than the parent compound, former catchment scale studies of substance release and flushing effects focused only on the parent compound. In this study, four river points were sampled in the Hula Valley, Israel, and samples were analyzed in the lab for chlorpyrifos (CP) and endosulfan residues (including transformation products; TPs). Sampling results of the first rainfall in autumn 2009 identified a strong release of most substances to the rivers. First flush effects of these substances were assessed regarding the risk for drinking water supply and ecology, like fresh water invertebrates and fish. Although, these substances were found in Jordan River water during the first significant rainfall the observed levels are below international drinking water guideline values with no adverse effects on human health in the region. However, the observed CP and chlorpyrifos oxon (CPO) levels are above the acute toxicity for fresh water invertebrates and fish. The study shows that the Hula Valley was an important source of pesticides and TPs at the Upper Jordan River basin and that substance flushing is extremely important for pesticides-monitoring campaigns.