► Flow regime in a south Ontario dolostone aquifer is investigated using a range of field methods. ► Two main levels of karst-enhanced permeability could be identified in the dolostone. ► The karst flow system became non-functional after the aquifer confinement by glacial sediments. ► Groundwater flow and transport are mainly controlled by the fractured network. The Silurian dolostone bedrock in Ontario, Canada, is a broad 400 km long swath northward from Niagara Falls through the Bruce Peninsula that represents an important water source for municipal, industrial, and agricultural uses. Where the Quaternary overburden is thin or absent, karst is common. This study concerns an urban area where the dolostone aquifer is 100 m thick beneath up to 50 m thick Quaternary deposits and where karst features identified by borehole information are common. Hydraulic tests show moderate to large bulk rock hydraulic conductivity and rock core tests indicate much smaller matrix hydraulic conductivity than the bulk rock values. Therefore, the aquifer is essentially a dual permeability, fully saturated system in which conduits occur within a network of ubiquitous extensive, horizontally- and vertically-interconnected fractures. Karst features are concentrated in a thin zone at the top-of-rock, likely representing former epikarst, and also in a thicker zone in the middle of the aquifer. Some pumping test results and large yields of some municipal wells are consistent with conduit occurrences. However, atmospheric tritium, distributed-source contamination (Cl − , NO 3 - ), and a point-source pesticide plume (metolachlor) show detailed concentration distributions lacking influence of flow in conduits. Detailed hydraulic head profiles also show no influence of conduit flow. This study shows that when designing monitoring networks for groundwater quality and source water protection in similar contexts, locating conduits is not necessary because contaminant distributions are governed by the combined influences of the rock matrix, fractures and conduits, the hydraulic boundary conditions, and the interconnected fracture network with only minimal conduit effects. Prior to glaciations, an integrated karstic aquifer could develop with flow controlled by conduits; however, this original, converging flow system became non-functional when the Quaternary sediments drastically modified the boundary hydrologic conditions and the head distribution.