Although regenerating forests make up an increasingly large portion of humid tropical landscapes, little is known of their water use and effects on streamflow (Q). Since the 1950s the island of Puerto Rico has experienced widespread abandonment of pastures and agricultural lands, followed by forest regeneration. This paper examines the possible impacts of these secondary forests on several Q characteristics for 12 mesoscale catchments (23-346 km2; mean precipitation 1720-3422 mm yr-1) with long (33-51 yr) and simultaneous records for Q, precipitation (P), potential evaporation (PET), and land cover. A simple spatially-lumped, conceptual rainfall-runoff model that uses daily P and PET time series as inputs (HBV-light) was used to simulate Q for each catchment. Annual time series of observed and simulated values of four Q characteristics were calculated. A least-squares trend was fitted through annual time series of the residual difference between observed and simulated time series of each Q characteristic. From this the total cumulative change (Â) was calculated, representing the change in each Q characteristic after controlling for climate variability and water storage carry-over effects between years. Negative values of Â were found for most catchments and Q characteristics, suggesting enhanced actual evaporation overall following forest regeneration. However, correlations between changes in urban or forest area and values of Â were insignificant (P ≥ 0.389) for all Q characteristics. This suggests there is no convincing evidence that changes in the chosen Q characteristics in these Puerto Rican catchments can be ascribed to changes in urban or forest area. The present results are in line with previous studies of meso-and macro-scale (sub-)tropical catchments, which generally found no significant change in Q that can be attributed to changes in forest cover. Possible explanations for the lack of a clear signal may include errors in the land cover, climate, Q, and/or catchment boundary data; changes in forest area occurring mainly in the less rainy lowlands; and heterogeneity in catchment response. Different results were obtained for different catchments, and using a smaller subset of catchments could have led to very different conclusions. This highlights the importance of including multiple catchments in land-cover impact analysis at the mesoscale. © 2013 Author(s).