The dual targeting ability of a variety of proteins from Physcomitrella patens, Oryza sativa and Arabidopsis thaliana were tested to determine when dual targeting arose and to what extent it is conserved in land plants. Overall the targeting ability of over 80 different proteins from rice and Physcomitrella representing 42 dual-targeted proteins in Arabidopsis were tested. It was found that dual targeting arose early in land plant evolution as it was evident in many cases with Physcomitrella proteins, which were conserved in rice and Arabidopsis. Furthermore, it was evident that the acquisition of dual targeting ability is still occurring, evident in Physcomitrella, as well as rice and Arabidopsis. The loss of dual targeting ability appears rare, but does occur. Ascorbate peroxidase represents such an example, following gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Whilst dual targeting was generally observed to be conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore it appears that small changes in the targeting signal can result in a loss (or gain) of dual targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual targeting ability. The acquisition of dual targeting ability also appears to be coordinated between proteins, Mia40, a protein involved in oxidative folding in mitochondria and peroxisomes, provides an example where acquisition of dual targeting is accompanied by the dual targeting of substrate proteins.