A comparative and systematic analysis of the different experimental conditions used in Trypanosoma cruzi-macrophage interaction assays is presented. A pH range from 6.2 to 6.9 is favorable for parasite invasion, as is the use of Dulbecco's Modified Eagle Medium (DMEM). The washing procedures used in purification protocols also enhance the parasites' ability to penetrate macrophages. However, the main factor affecting the quantitative parameter of this in vitro infection, regardless of the parasite: cell ratio, is the number of macrophages provided to the parasites. These results, reviewed and compared with the corresponding literature, strongly suggest that macrophage surface areas and/or receptors are the limiting factors for optimal quantitative determination of the interaction of T. cruzi in vitro.