We experimentally investigated the equilibrium conformation and dynamics of single DNA molecules in slitlike nanochannels. We measured the in-plane radius of gyration (R-parallel to), diffusivity (D), and longest relaxation time (tau) of lambda-DNA (48.5 kbp) as functions of the slit height using fluorescence microscopy. Our results show that the in-plane radius of gyration increases monotonically with decreasing slit height, in contrast to results from Bonthuis et al. (15)but in agreement with our simulations and those of other groups. In strong confinement (slit height <100 nm), the scaling of D, tau, and R-parallel to with slit height does not show an evident change, suggesting that the transition from the de Gennes regime to the Odijk regime is gradual and broad.