To analyze morphologic and physiological properties of olfactory interneurons in the honeybee, Apis mellifera, antennal lobe (AL) neurons were intracellularly recorded and subsequently labeled with Neurobiotin. Additional focal injections were carried out with cobalt hexamine chloride and dextran fluorescent markers. Olfactory interneurons (projection neurons, PNs) project by means of five tracts, the lateral, the median, and three mediolateral antennocerebral tracts (l-, m-, and ml-ACT, respectively) to the mushroom bodies (MBs) and the protocerebral lobe (PL) of the ipsilateral protocerebrum. Uniglomerular PNs of the m- and l-ACT receiving input from a single glomerulus of the AL also arborize in different regions of the AL. The vast majority of l-ACT innervate the T1 region, whereas m-ACT neurons arborize exclusively in the T2, T3, and T4 regions (T1-4 : AL projection area of sensory cells from the antennae). In the calyces of the MB, uniglomerular PNs form varicosities in the basal ring and the lip region. Individual neurons of both types exhibit unequal innervation within and between the two calyces. In addition, m-ACT fibers ramify more densely within the lip neuropil and show a higher incidence of spine-like processes than l-ACTs. In the PL, l-ACTs arborize exclusively within the lateral horn, whereas some m-ACT neurons innervate a broader region. Multiglomerular neurons of the ml-ACT leave the AL by means of three subtracts (ml-ACT 1–3). Two different types can be distinguished according to their protocerebral target areas: ml-ACTs projecting to the lateral PL (LPL) and to the neuropil around the α-lobe (tracts 2 and 3) and neurons projecting only to the LPL (tract 1). Intracellular recordings indicate that both l- and m-ACT neurons respond to general odors but with different response properties, indicating that odor information is processed in parallel pathways with different functional characteristics. Just like m-ACT neurons, ml-ACT neurons respond to odors with complex activity patterns. Bilateral interneurons, originating in the suboesophageal ganglion, connect glomeruli of both AL, and send an axon through the m-ACT in each hemisphere of the brain, terminating in the lip region of the calyces. These neurons respond to contact chemical stimuli. J. Comp. Neurol. 437:363–383, 2001. © 2001 Wiley-Liss, Inc.