The antennal lobes (ALs) are the primary olfactory centers in the insect brain. In the AL of the honeybee, olfactory glomeruli receive input via four antennal sensory tracts (T1–4). Axons of projection neurons (PNs) leave the AL via several antenno-cerebral tracts (ACTs). To assign the input–output connectivity of all glomeruli, we investigated the spatial relationship of the antennal tracts and two prominent AL output tracts (medial and lateral ACT) mainly formed by uniglomerular (u) PNs using fluorescent tracing, confocal microscopy, and 3D analyses. Furthermore, we investigated the projections of all ACTs in higher olfactory centers, the mushroom-bodies (MB) and lateral horn (LH). The results revealed a clear segregation of glomeruli into two AL hemispheres specifically supplied by PNs of the medial and lateral ACT. PNs of the lateral ACT innervate glomeruli in the ventral-rostral AL and primarily receive input from T1 (plus a few glomeruli from T2 and T3). PNs of the medial ACT innervate glomeruli in the dorsal-caudal hemisphere, and mainly receive input from T3 (plus a few glomeruli from T2 and T4). The PNs of the m- and l-ACT terminate in different areas of the MB calyx and LH and remain largely segregated. Tracing of three mediolateral (ml) ACTs mainly formed by multiglomerular PNs revealed terminals in distinct compartments of the LH and in three olfactory foci within the lateral protocerebrum. The results indicate that olfactory input in the honeybee is processed via two separate, mainly uPN pathways to the MB calyx and LH and several pathways to the lateral protocerebrum. J. Comp. Neurol. 499:933–952, 2006. © 2006 Wiley-Liss, Inc.