There is debate whether interval carcinomas differ from screen-detected tumours biologically. In this study, clinico-pathological parameters and the expression of well-validated biological markers were compared between 'true' interval carcinomas and screen-detected/missed carcinomas hypothesising that 'true' interval carcinomas show a more aggressive biological behaviour. The study group consisted of 92 consecutive postmenopausal women attending the breast screening programme and presenting with an invasive ductal carcinoma. All screening mammograms were re-reviewed. Sixteen patients had a 'true' interval carcinoma. Seven carcinomas were missed at screening, but detected on re-reviewing of the screening mammogram. Radiological characteristics were assessed from diagnostic mammograms. Data on patient- and tumour characteristics and follow-up data were recorded from hospital records. Median follow-up was 61 months. Immunohistochemistry for ER, PR, Her2/neu and p53 was performed on TMA sections. Univariate and multivariate logistic regression analyses were performed. In univariate analysis, 'true' interval carcinomas were significantly larger (odd ratios (OR) 7.2, 95% CI 1.8-28.1) and less frequently ER (OR 0.3, 95% CI 0.1-0.9) and PR (OR 0.3, 95% CI 0.1-1.0) positive. In multivariate analysis, 'true' interval carcinoma was independently associated with larger tumours (OR 7.0, 95% CI 1.4-36.2). A trend toward ER negativity was found (OR 0.3, 95% CI 0.1-1.1). 'True' interval carcinomas showed a trend toward a decreased relapse-free survival (HR 1.7 95% CI 0.9-3.1). Although 'true' interval carcinomas were significantly larger than screen-detected/missed interval carcinomas, it remains challenging to observe parameters that determine this difference between 'true' interval carcinomas and screen-detected lesions.