The infection of foetal thymus with coxsackievirus B4 (CV-B4) E2 has been studied ex vivo by using CD-1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV-B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive- and negative-stranded viral RNA in cells by using a real-time quantitative RT-PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post-infection (p.i.). Staining of tissue sections with an anti-cytokeratin antibody and haematoxylin-eosin showed that CV-B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock- and virus-infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7-amino-actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV-B4 E2-infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3(-)CD4(-)CD8(-) thymocytes, and a decrease in the percentage of immature CD3(-)CD4(+)CD8(+) cells, together with an increase in the percentage of mature CD3(+)CD4(+) and CD3(+)CD8(+) cells. These data show that CV-B4 E2 disturbs T-cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T-cell differentiation.