Although injured neurons of inferior colliculus (IC) display a robust axonal outgrowth through a lesion site at postnatal day six (P6) in vitro, and are capable to re-innervate their target cells, injured neurons from P10 IC are unable to regenerate their axons across the lesion site. This axonal regenerative failure has been attributed to an increase of expression of inhibitory molecules in endogenous tissue, during development. As a first step to identify such inhibitory molecules, the present study reports the isolation of molecules differentially expressed in the IC during development. A two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on IC tissue between P6 and P10 stages. One hundred cDNAs from P6 (P6-P10) and 200 cDNAs from P10 (P10-P6)-subtracted libraries were randomly sequenced. A dot-blot screening of sequenced cDNAs revealed the differential expression for the majority of these cDNAs at their respective developmental stage. Then, the analysis of sequenced clones showed that P6 library was highly enriched in molecules expressed early in the development, such as GAP43 or vimentin proteins. By contrast, the P10 library contained mostly molecules expressed at later stages of development in the central nervous system, such as myelin-related proteins. Our results show that SSH is a suitable method for identifying differentially expressed genes in the developing IC. In addition, these results provide a foundation for further studies dealing with molecules involved in the IC development before and at the onset of hearing, some of which being probably involved in the axonal outgrowth mechanism.