Subsurface flow from various portions of a soil profile on a steep, forested hillslope was evaluated by two sets of step-change miscible displacement tests at different application rates and antecedent hydrologic conditions. Solutions of NaCl (1000 mg L−1 Cl−) were applied at steady state rates (equivalent to 20 and 30 mm h−1 of standing water over the entire plot area) using a line irrigation source located 1.5 m upslope (lateral distance) from an excavated soil pit. Subsurface flow and tracer breakthrough from five portions (the organic-rich soil layer including macropores, the mineral soil matrix, and three groups of macropores in the mineral soil layer) of the soil profile were individually measured and analyzed using a convective-dispersive model. Matrix flow dominated discharge from the soil pit during tracer tests (70–93% of total discharge). However, during wet periods with upslope drainage, macropores (including organic-rich soil) contributed proportionally more flow than during periods when upslope drainage was minimal. Outflow from macropores during the test with wet antecedent conditions had lower Cl− concentrations than drainage from the soil matrix, suggesting dilution in macropores from upslope drainage. Effective pore volumes calculated for the flow-averaged breakthrough data from the entire profile were much less (<40%) than the estimates (measured by tensiometers) of total volume of pore water, suggesting that preferential flow significantly contributed to subsurface transport of tracer. The pore volume for the entire profile increased only slightly with increasing application rate; however, the relative proportions of pore volumes calculated for individual portions varied proportionally to antecedent hydrologic conditions. These changes are attributed to the expansion of individual macropores with surrounding soil and the lateral extension of macropore networks during wetter conditions.