The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen. To examine cid and lrg promoter activity during biofilm development, fluorescent reporter fusion strains were constructed and grown in a BioFlux microfluidic system, generating time-lapse epifluorescence images of biofilm formation, which allows the spatial and temporal localization of gene expression. Consistent with cid induction under hypoxic conditions, the cid::gfp fusion strain expressed green fluorescent protein predominantly within the interior of the tower structures, similar to the pattern of expression observed with a strain carrying a gfp fusion to the hypoxia-induced promoter controlling the expression of the lactose dehydrogenase gene. The lrg promoter was also expressed within towers but appeared more diffuse throughout the tower structures, indicating that it was oxygen independent. Unexpectedly, the results also demonstrated the existence of tower structures with different expression phenotypes and physical characteristics, suggesting that these towers exhibit different metabolic activities. Overall, the findings presented here support a model in which oxygen is important in the spatial and temporal control of cid expression within a biofilm and that tower structures formed during biofilm development exhibit metabolically distinct niches.