Abstract Spraydrift and edge-of-field runoff are important routes of pesticide entry into streams. Pesticide contamination originating from spraydrift usually resides in the water phase, while pesticides in contaminated runoff are to a large extent associated with suspended particles (SPs). The effects of two organophosphorous insecticides (OPs), chloropyrifos (CPF) and azinphos-methyl (AZP), on acetylcholinesterase (AChE) activity in rainbow trout were compared between two exposure scenarios, simulating spraydrift- and runoff-borne contamination events in the Lourens River (LR), Western Cape, South Africa. NOECs of brain AChE inhibition, determined after 1 h of exposure followed by 24 h of recovery, were 0.33 μg l −1 for aqueous CPF, 200 mg kg −1 for SP-associated CPF and 20 mg kg −1 for SP-associated AZP (at 0.5 g l −1 SP). The highest aqueous AZP concentration tested (3.3 μg l −1) was without significant effects. Previously reported peak levels of aqueous CPF in the LR (∼0.2 μg l −1) are close to its NOEC (this study), suggesting a significant toxicological risk to fish in the LR. By contrast, reported levels of SP-associated OPs in the LR are 20–200-fold lower than their NOECs (this study). In a comparative in situ study, trout were exposed for seven days at agricultural (LR2, LR3) and upstream reference (LR1) sites. No runoff occurred during the study. Brain AChE was significantly inhibited at LR3. However, OP levels at LR3 (CPF 0.01 μg l −1; AZP 0.14 μg l −1) were minor compared to concentrations having effects in the laboratory (see above). Additionally, muscle AChE activity was significantly higher in caged trout from LR1 than in animals maintained in laboratory tanks.