Bispecific T-cell engager (BiTE) molecules are designed to engage and activate cytotoxic T cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. Methods: To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with the PET isotope Zr-89 and studied in immunocompetent and immunodeficient mouse models. Results: PET images and ex vivo biodistribution in immunocompetent mice with [Zr-89]Zr-DFO-N-suc-muS110, targeting mouse CD3 (dissociation constant [K-D], 2.9 nM) and mouse epithelial cell adhesion molecule (EpCAM; K-D, 21 nM), and with [Zr-89]Zr-DFO-N-suc-hyS110, targeting only mouse CD3 (K-D, 2.9 nM), showed uptake in the tumor, spleen, and other lymphoid organs, whereas the human-specific control BiTE [Zr-89]Zr-DFO-N-suc-AMG 110 showed similar tumor uptake but lacked spleen uptake. [Zr-89]Zr-DFO-N-suc-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of nonradiolabeled muS110 to immunocompetent mice, Zr-89-muS110 uptake in the spleen and other lymphoid tissues decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of [Zr-89]Zr-DFO-N-suc-muS110 and [Zr-89]Zr-DFO-N-suc-hyS110 with CD3-positive T cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T cells. Conclusion: [Zr-89]Zr-DFO-N-suc-muS110 biodistribution is dependent mainly on the T-cell-targeting arm, with a limited contribution from its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs, as the results might have implications for their respective drug development and clinical translation.