Respiratory tract dendritic cells (RTDC) form a contiguous subepithelial network within the nasorespiratory tract bridging innate and acquired immunity and have been implicated in nasal mucosal tolerance induction. Discrepancies exist between isolation techniques with respect to phenotype and function of RTDC. Therefore, the aim of this study was to modify previous methods to provide a consistent isolation method whilst maintaining good cell viability and enriched cell numbers so as to facilitate further phenotype and functional studies of murine RTDC. RTDCs isolated by enzyme digestion, Percoll density gradient centrifugation and overnight GM-CSF culture followed by MACS separation retain an archetypical immature dendritic cell phenotype, characterised by MHCII(low) CD40(neg) CD86(neg) CD80(neg) CD11c(low) cell surface expression. Splenic-derived DC (SDC) isolated conformed to a day 1 in vitro phenotype; MHCII(low) CD40(neg) CD86(low) CD80(neg) CD11c(low) but can further mature phenotypically in vitro. Both RTDC and SDC processed and presented antigen efficiently to T cells in vitro. Using such modified isolation procedures for RTDCs, we have developed a consistent method of RTDC enrichment, which maintains the immature phenotype and functional antigen presenting capability.