Purpose Endometriosis is characterised by inflammation, fibrosis and scarring, all processes known to involve transforming growth factor-β1 (TGFB1). We examined the expression of genes involved in TGFB1 signalling in epithelium and stroma from eutopic versus ectopic endometrium in women with endometriosis. Methods Eutopic and ectopic endometrium was collected during laparoscopy from women with endometriosis (n = 4 proliferative, n = 7 secretory). Laser capture microdissection was used to collect glandular epithelium and CD10-positive stroma from ectopic lesions and eutopic endometrium. Quantitative real-time RT-PCR was used to examine TGFB1, SMAD family member-2 (SMAD2), SMAD3 and SMAD4 mRNA. Expression of mRNA was relative to an RNA-spike. B2M and RPL13A mRNA, and 18S rRNA were also examined; however, expression varied between eutopic and ectopic sites making them unsuitable housekeeping genes in this model. Results TGFB1, SMAD3 and SMAD4 mRNA expression was significantly higher in eutopic versus ectopic glandular epithelium; SMAD3 expression was significantly higher in secretory versus proliferative phase eutopic epithelium. TGFB1 expression was significantly higher in eutopic versus ectopic stroma. Conclusions Variations in TGFB1, SMAD3 and SMAD4 mRNA expression in eutopic relative to ectopic tissues are consistent with a specific function in eutopic endometrium not replicated in lesions. We suggest that TGFB1-mediated activity is altered in ectopic lesions relative to eutopic endometrium in women with endometriosis, which may affect lesion maintenance. Further research examining the functional consequences of TGFB1 signalling variations is required, including extension of these studies to women without the disorder. Further studies must also carefully consider their housekeeping method and its impact on data and conclusions.