The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::beta-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.