Targeted aequorins (CYT-AEQ, NUC-AEQ, and MT-AEQ) were used to measure Ca2+ concentrations within organelles of live trypanosomes. We determined that the nuclear envelope is a slight barrier to the free diffusion of Ca2+. This situation was especially evident when Ca2+ influx across the plasma membrane was stimulated with 200 nM melittin ([Ca2+]cyt = 1.2 +/- 0.4 microM and [Ca2+]nuc = 0.85 +/- 0.15 microM). By contrast, the ionophores nigericin (2.7 microM) or monensin (2 microg/ml) were used to induce Ca2+ efflux from the acidic storage compartment. Small transient elevations in [Ca2+]cyt were observed (peaking at 660 +/- 200 and 580 +/- 120 nM, respectively). Parallel and equivalent changes in [Ca2+1]nuc were recorded. Active accumulation of Ca2+ into the nucleus was not observed. Nigericin or monensin did not disrupt mitochondrial Ca2+ transport in vivo. Instead, the mitochondrion actively sequestered large quantities of Ca2+ in the presence of these ionophores, with peak values of 2.7 +/- 1.4 and 4.4 +/- 1.1 microM, respectively. Overall, these data demonstrate that significant quantities of Ca2+ enter the nucleus following influx across the plasma membrane or following efflux from an intracellular acidic storage compartment. However, the magnitude of change for [Ca2+]cyt and [Ca2+]nuc is small compared to the total amount of exchangeable Ca2+ since the majority of released Ca2+ is actively sequestered by the mitochondrion.