Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.