Using total internal reflection, fluorescence microscopy (TIRFM), we have observed the directed motion of 20 nm probe particles on specific regions of surfaces that exhibited strong gradients of hydrophobicity. Patterned surfaces were prepared by selective photodegradation (using a contact photomask) of a hydrophobically modifed fused silica surface. The lateral distribution of hydrophobicity was characterized in situ using the selective affinity of amphiphilic probes (i.e., hydrophobic interaction microscopy). Probe particles were observed to move unidirectionally from regions of lower to higher to hydrophobicity over distances of similar to 1 mu m when the hydrophobicity gradient was greater than d(cos theta)/dx = 0.05 +/- 0.02 mu m(-1), where theta is the water contact angle on the bare surface. Only adsorption events were observed oil energetically homogeneous surface regions.