In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.