The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly (D, L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8+ and CD8− DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after sc injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8− DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8+ DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8− DCs and MΦs.