To establish if the cell adhesion molecule L1 could promote neurite outgrowth of septal neurons, L1-positive substrates were prepared by genetically modifying 3T3 fibroblasts with a retroviral vector encoding human L1 under the control of a negative tetracycline-regulatory system. In several clones of L1-transfected fibroblasts, L1 expression at the cell surface was prominent and efficiently regulated by doxycycline, a tetracycline analogue. In co-culture of septal neurons and fibroblasts, a two-dimensional fractionator probe provided systematic random sampling of the neurites to be measured. Septal neurons, isolated at embryonic Day 17, were found to express L1 in vitro and to extend significantly longer neurites when plated on L1-expressing fibroblasts compared to control fibroblasts. The neurite outgrowth-promoting effect of L1 was inhibited after a doxycycline treatment, which specifically suppressed L1 expression from the modified fibroblasts. The findings that septal neurons at embryonic Day 17 in vitro express L1 and respond to L1-modulation suggest that this molecule is involved in development of the septohippocampal pathway. © 2004 Wiley-Liss, Inc.