A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment.