Mycobacterium avium subspecies paratuberculosis (Map) is an intracellular pathogen of monocytes and macrophages. Increased cytosolic calcium is a early event following phagocytosis which is involved in multiple signaling cascades including activation of kinases, cytoskeletal alterations, and phagolysosome development. Our laboratory is developing an assay for detecting changes in cytosolic calcium concentrations in macrophages using flow cytometric techniques. The hypothesis of this study is that cytosolic calcium levels do not significantly increase following uptake of Map into J774 cells. We prepared suspension cultures of J774 macrophages and incubated them with the fluorescent calcium indicator Fluo-4 (Molecular Probes, Eugene, OR) for 20 minutes at 37ºC. Fluo-4 can cross cell membranes and after binding to cytosolic calcium is strongly fluorescent. By flow cytometry we determined the baseline level of Fluo-4 fluorescence in noninfected cells. Next, we incubated cells with either the ionophore ionomycin, Map, or zymosan A, and monitored Fluo-4 fluorescence for 6-10 minutes. As expected, following the addition of ionomycin there was a consistent increase in Fluo-4 fluorescence. We did not demonstrate a significant increase in Fluo-4 fluorescence after addition of Map to the J774 cell suspensions; however, we did detect increases in Fluo-4 signal following addition of zymosan A. These results of this study suggest that following uptake of Map into J774 cells there is no significant change in the cytosolic calcium.