Field observations of runoff generation in pristine montane cloud forests are scarce. However, this knowledge is important for a sustainable natural resources management. Here we report results of a study carried out in the San Francisco River basin (75,3 km2) located on the Amazonian side of the Cordillera Real in the southernmost Andes of Ecuador. The basin is mainly covered with cloud forest, sub-páramo, pasture and ferns. A nested sampling approach was used for the collection of stream water samples and discharge measurements in the main tributaries and outlet of the basin. Additionally, soil and rock water samples were collected. Weekly to biweekly water grab samples were taken at all stations in the period April 2007 to November 2008. Hydrometric, mean residence time and mixing model approaches allowed identifying the main hydrological processes that control the runoff generation in the basin. Results clearly reveal that flow during dry conditions mainly consists of lateral flow through the C-horizon and cracks in the top weathered bedrock layer. The data shows that all catchments have an important contribution of this deep water to runoff, no matter whether pristine or deforested. During normal to low precipitation intensities, when antecedent soil moisture conditions favor water infiltration, vertical flow paths to deeper soil horizons with subsequent lateral sub-surface flow contributes most to streamflow. Under wet conditions in forested catchments streamflow is controlled by near-surface lateral flow through the organic horizon, and it is unlikely that Horton overland flow occurs during storm events. By absence of the litter layer in pasture streamflow under wet conditions primarily originates from the rooted surface layers and the A horizon, and Hortonian overland flow during extreme events.