In this paper, we study the variation of the thickness of patterned microchannel features in photoresist (PR) by two-step photolithography. The final PR thickness is determined by the thickness and width of the predefined PR pattern in the first-step lithography and the thickness of the spin-coated PR film in the second-step lithography. Thickness variation is demonstrated to be an important consideration within the critical pattern width that the capillary pressure is still dominant for forming the PR film thickness. With this mechanism of two-step photolithography, we demonstrate its ability to form multilevel channel features and its use to fabricate a cross-flow filter with two different pore sizes as a microfluidic application. (C) 2010 American Institute of Physics. [doi:10.1063/1.3517230]