Tenascin-C (TN) is an extracellular matrix protein that is expressed at low levels in normal adult tissue but is highly expressed around many tumors including breast carcinoma. TN exists as multiple isoforms generated through alternative splicing, and these isoforms have different effects on cell growth and migration. This study has analyzed in detail the pattern of TN isoform expression in benign, preinvasive, and invasive breast lesions using reverse transcription-PCR and Southern blotting. Significant differences in the profile of TN isoforms were identified. Although all tissues expressed the fully truncated TN, expression of two additional isoforms, one containing exon 16 (TN16) and one containing both exons 14 and 16 (TN14/16), were significantly associated with the invasive phenotype (P < 0.001). A subset of ductal carcinoma in situ (DCIS) cases were also found to express these isoforms, which may be indicative of a high risk of invasion in these lesions. Expression of these isoforms correlated with the presence of TN protein in the stroma in place of or in addition to basement membrane TN. Immunohistochemistry and in situ hybridization confirmed the production of exon 14-containing higher molecular weight isoforms by stromal fibroblasts in malignant tissue and both periductal fibroblasts and residual myoepithelial cells in DCIS. Although no evidence of tumor cell synthesis of TN was detected in the tissues, two highly invasive breast cancer cell lines (MDA-MB 231 and MDA-MB 468) were found to produce TN in contrast with tumor cells with a lower invasive capacity (MCF-7 and T47D). These results demonstrate for the first time that specific TN isoforms are expressed in invasive breast carcinomas and that these isoforms are identified in a subset of DCIS and suggest that detection of TN16 and/or TN14/16 may be used as a predictor for invasion. Functional studies are now essential to establish the effect of these isoforms on tumor behavior and evaluate whether they will provide appropriate targets for therapeutic intervention.