Enhancers play an important role in chromatin opening but the temporal relationship between enhancer activation and the generation of an accessible chromatin structure is poorly defined. Recombination enhancers are essential for chromatin opening and subsequent V(D)J recombination at immunoglobulin loci. In mice, the kappa light chain locus displays an open chromatin structure before the lambda locus yet the same proteins, PU.1/PIP, trigger full enhancer activation of both loci. Using primary B cells isolated from distinct developmental stages and an improved method to quantitatively determine hypersensitive site formation, we find the kappa and lambda recombination enhancers become fully hypersensitive soon after transition to large and small pre-B-II cells, respectively. This correlates strictly with the stages at which these loci are activated. Since these cells are short-lived, these data imply that there is a close temporal relationship between full enhancer hypersensitive site formation and locus chromatin opening.