The cardiac conduction system (CCS) consists of the sinoatrial and atrioventricular nodes, the bundle of His, the bundle branches and the Purkinje fibres (PF). The PF form the terminal portion of the ventricular conduction system and provide a rapid conduction pathway. They have been shown to have spontaneous diastolic depolarization, a more negative plateau potential than ventricular muscle and a susceptibility to the production of early afterdepolarizations. The PF have been linked to a number of ventricular arrhythmias including torsade de pointes arrhythmias associated with long-QT syndrome. The whole-mount immunohistochemical method employing middle neurofilament as a marker of the CCS was used to map the anatomical distribution of the bundle branches and the free running left and right Purkinje fibres (LPF and RPF) in the rabbit heart. The left bundle branch (LBB) appeared as a ribbon like structure composed of many fine fibres (perhaps only one myocyte wide) running in parallel on the septal surface. The fine fibres formed denser strands before branching into a network of fine fascicles that covered the left ventricular free wall. The right bundle branch appeared to be narrower than LBB and ran over the septal surface. It had a number of fascicles branching from it along its length. These strands extended across the ventricle and formed a dense network on the right ventricular free wall. Quantitative RT-PCR was used to analyse the expression of various transcripts in eight rabbits. ~30 transcripts for ion channels, connexins, Ca2+-handling proteins and cellular markers were investigated in the LPF, RPF, right atrium (RA) and left ventricle (LV). The results show that the PF have an ion channel expression profile distinct from that of the working myocardium. In the LPF and/or RPF, there was a significantly higher expression (versus LV) of Nav1.1, HCN1, HCN4 and NFM mRNAs and a significantly lower expression of Cav1.2, KvLQT1, ERG, KChIP2, SUR2, Cx45, RYR2, SERCA2a and NCX1 mRNAs. There was also a tendency for a higher expression of Kir3.1, TWIK-1 and Cx40 mRNAs and a lower expression of Kv1.4, Kir2.1, Kir6.2, RYR3 and NCX1 mRNAs in the PF. The expression in the free running PF was also distinct from that in the RA: in the LPF and/or RPF there was a significantly higher expression of NFM mRNA and a significantly lower expression of HCN4, Kir3.1, KChIP2, Cx45 and ANP mRNAs. There was also a tendency for a higher expression of Nav1.1, Kv4.2, Kv4.3, TWIK-1, Kir2.1 and minK mRNAs and a lower expression of Navβ1, Kir6.2, SUR2, Cx40 and SERCA2a mRNAs in the PF. We have demonstrated that the free running PF form a complex asymmetrical network in the left and right ventricular chambers of the rabbit heart. We have also shown that the free running PF have a unique pattern of expression of ion channels, connexins, Ca2+ -handling proteins and cellular markers.