Emplacement of highways and railroads has altered natural hydrologic systems by influencing surface-water flow paths and biotic communities in Mojave National Preserve. Infiltration experiments were conducted along active and abandoned channels to evaluate changes in hydrology and related effects on plant water availability and use. Simulated rainfall infiltration experiments with vegetation monitoring were conducted along an active channel upslope and a comparable abandoned channel down slope of the transportation corridor. We also conducted 90 single-ring, ponded infiltration experiments in adjacent channels to evaluate field-saturated hydraulic conductivity and particle size distributions. The abandoned channels are still morphologically evident, but are disconnected from runoff sources at higher elevations. Infiltration test results show that water infiltrates twice as fast in the active channels. Excavation showed weak soil development with fewer plant roots beneath the abandoned channel. SEM analysis on surface samples showed the presence of cyanobacteria only in abandoned channels. Plants up to three meters away from both channels showed physiological responses to channel water applied in a simulated pulse of rain. The response was short-lived and less pronounced for plants adjacent to the abandoned channel, whereas those adjacent to the active channel showed responses up to two months after the pulse. These responses may explain observed lower plant densities and fewer deep-rooted species along abandoned channels compared to active channels. We infer that the deeper-rooting plants are more abundant where they are able to take advantage of the increased soil-water storage resulting from greater infiltration and flow frequency in active stream channels.