We analysed the expression of Tic20 in Pisum sativum and Arabidopsis thaliana by quantitative RT-PCR, and compared it directly with the expression of Tic110 in both organisms. Furthermore, semi-quantitative immunoblot analyses revealed the absolute amounts of Tic20 and Tic110 in chloroplast envelopes. Moreover, we showed that Tic20 and Tic110 are not part of a mutual complex in isolated pea IE. After the successful expression and purification of Tic20 we were able to experimentally verify its predicted ahelical structure and Nin-Cin topology. Finally, we report for the first time that Tic20 forms a cation selective channel when reconstituted into liposomes.Due to errors in the annotation of AtTic20-I, currently available Affymetrix micro-arrays do not contain specific oligonucleotides for this isoform and therefore cannot be used to investigate the expression levels of AtTic20-I.