The advance of axonal tract-tracing has revolutionized neuroscience in the past three decades1. The elementary purpose of neuronal tract-tracing is to chart anatomical connections within the nervous system providing useful information on afferent and efferent connectivity in the brain. Biocytin is a classical neuroanatomical tract-tracer that is taken up by neurons and transported in both antero- and retrograde directions. Our aim was to develop non-toxic, efficient neuronal tracers that allow both, in vivo brain connectivity studies by means of MRI and postmortem-microscopic investigation in fixed tissue, in the same experimental animal. We have designed five novel biocytin-based neuroanatomical tract-tracers (L1-L5). In newly modified-biocytin (L1), propylamine is linked to amide of biocytin to make it tert-amide, which is stable to biotinidase degradation. The propylamine of L1was used as a linker to link, FITC as a fluorescent moiety (L2) or Gd-DOTA as MR detectable part (L3). L4 has an amide linkage between amine of GdDO3A-EA and carboxylate of biocytin while L5 consists of a novel precursor based on serine containing Gd-DO3A. This precursor has an amine and a carboxylate group available for coupling of biotin and l-lysine. In vivo histological experiments with L1 demonstrated an increased molecular stability compared with biocytin and excellent neuronal tract-tracing capabilities. In vitro efficiency of cell internalization of L2 into N18 neuroblastoma cells was demonstrated by fluorescence microscopy. In vitro MRI of L3-L5 with increasing concentrations of avidin were performed at 7T. The increase in R2 for L3-L5 (300%-100% respectively) demonstrated strong binding of all tracers in the pocket of tetrameric avidin through biotin. The above studies and preliminary results reveal a new strategy for neuroanatomical tract-tracing, which combines the powerful spatial resolution of the conventional microscopic techniques with the whole brain tri-dimensional coverage and in vivo applicability of MRI. 1. Progress in Neurobiology 62 (2000), 327.