Abstract Introduction Tenascin-C (TNC) is a large extracellular matrix glycoprotein that shows prominent stromal expression in many solid tumours. The profile of isoforms expressed differs between cancers and normal breast, with the two additional domains AD1 and AD2 considered to be tumour associated. The aim of the present study was to investigate expression of AD1 and AD2 in normal, benign and malignant breast tissue to determine their relationship with tumour characteristics and to perform in vitro functional assays to investigate the role of AD1 in tumour cell invasion and growth. Methods Expression of AD1 and AD2 was related to hypoxanthine phosphoribosyltransferase 1 as a housekeeping gene in breast tissue using quantitative RT-PCR, and the results were related to clinicopathological features of the tumours. Constructs overexpressing an AD1-containing isoform (TNC-14/AD1/16) were transiently transfected into breast carcinoma cell lines (MCF-7, T-47 D, ZR-75-1, MDA-MB-231 and GI-101) to assess the effect in vitro on invasion and growth. Statistical analysis was performed using a nonparametric Mann-Whitney test for comparison of clinicopathological features with levels of TNC expression and using Jonckheere-Terpstra trend analysis for association of expression with tumour grade. Results Quantitative RT-PCR detected AD1 and AD2 mRNA expression in 34.9% and 23.1% of 134 invasive breast carcinomas, respectively. AD1 mRNA was localised by in situ hybridisation to tumour epithelial cells, and more predominantly to myoepithelium around associated normal breast ducts. Although not tumour specific, AD1 and AD2 expression was significantly more frequent in carcinomas in younger women (age ≤40 years; P < 0.001) and AD1 expression was also associated with oestrogen receptor-negative and grade 3 tumours (P < 0.05). AD1 was found to be incorporated into a tumour-specific isoform, not detected in normal tissues. Overexpression of the TNC-14/AD1/16 isoform significantly enhanced tumour cell invasion (P < 0.01) and growth (P < 0.01) over base levels. Conclusions Together these data suggest a highly significant association between AD-containing TNC isoforms and breast cancers in younger women (age ≤40 years), which may have important functional significance in vivo.