pS2–TFF 1 is expressed in breast cancers and has been investigated as a potential prognostic factor reflecting oestrogen dependence. The relationship to the expression of other trefoil peptides, human spasmolytic polypeptide (hSP–TFF 2) and intestinal trefoil factor (hITF/hPI.B–TFF 3) is documented here. Fifty-seven breast specimens were selected from surgical pathology archives and included five normal breasts (two lactating), seven benign proliferative lesions, 11 ductal carcinomas in situ (DCIS), three lobular carcinomas in situ(LCIS), 24 invasive ductal carcinomas (IDC), and seven invasive lobular carcinomas (ILC). The comparative distribution of trefoil mRNAs was assessed by in situ hybridization using 35S-labelled riboprobes and immunohistochemical staining for pS2–TFF 1 and hSP–TFF 2. pS2–TFF 1 and hITF/hPI.B–TFF 3 mRNA were focally present at low signal intensity in normal and benign breast. Both pS2–TFF 1 and hITF/hPI.B–TFF 3 were expressed in all DCIS, LCIS and ILC, and 21/24 IDC. Overall, expression patterns of pS2–TFF 1 and hITF/hPI.B–TFF 3 coincided, but hITF/hPI.B–TFF 3 mRNA was usually found in a greater proportion of cells. Expression of hSP–TFF 2 peptide or mRNA was not detected in any of these cases. MCF 7 breast carcinoma cells also expressed hITF/hPI.B–TFF 3 and pS2–TFF 1 mRNAs but not hSP–TFF 2. hITF/hPI.B–TFF 3 co-expression with pS2–TFF 1 may act as a prognostic factor, but also raises questions about the regulatory pathway for pS2–TFF 1 hITF/hPI.B–TFF 3. Trefoil factors have effects on cell motility and spreading in vitro, and co-expression of hITF/hPI.B–TFF 3 with pS2–TFF 1 could be functionally significant if they form a heterodimer or compete for receptor binding. Absence of hSP–TFF 2 expression may be of equal relevance to tumour cell biology.