We observed the free diffusion of individual 10 kbp (1/2)lambda (24.2 khp). and lambda (48.5 kbp) double-stranded deoxyribonucleic acid (DNA) molecules in fused-silica nanoslits with depths from 541 to 24 nm using epifluorescence video microscopy. Diffusivity. 1). scaled with nanoslit depth. It, according to D proportional to h(alpha), where a wits 0.47 +/- 0.05. 0.51 +/- 0.06, and 0.47 +/- 0.05 for 10 kbp (1/2)lambda, and lambda DNA, respectively, in disagreement with the value of two-thirds predicted by blob theory. We observed no chan(,e in scaling behavior for h less than the persistence length of DNA, as predicted by reflecting rod theory. The scaling of D with DNA length. N, followed D proportional to N-1, indicating hydrodynamic screening in accordance with Rouse dynamics. Our results establish these scaling exponents for smaller h and greater DNA confinement than previous studies.