Mouse thymocytes and erythrocytes form rosettes when incubated together at 4 degrees C. The frequency is much higher when the thymocytes and erythrocytes are MHC-identical. If the indolizidine alkaloid swainsonine (SW) is present during rosette formation at concentrations of 1 microgram/ml (5.7 microM) or greater, rosette formation between MHC-identical pairs is inhibited to levels comparable to those observed for MHC-different pairs; rosette formation by MHC-different pairs is not affected. This was confirmed by examining 17 different MHC-identical combinations (9 completely syngeneic and 8 differing in non-MHC genes) and 13 MHC-different combinations (3 of these identical everywhere except at MHC). A SW-inhibitable component of rosette formation was observed only when thymocyte and erythrocyte were completely identical at MHC. Thus F1-parent pairs behaved as if allogeneic, although both F1-F1 and parent-parent had a SW-inhibitable rosetting component. Similarly, inbred strains only partially MHC-identical (B10.BR-B10.A, B10.D2-B10.A) behaved as if allogeneic. The SW-inhibitable component of rosetting could be partially but significantly blocked by including monoclonal antibodies against Thy-1, and anti-CD4 plus anti-CD8 (together but not separately); anti-class-I-MHC produced some inhibition of marginal significance. Monoclonal antibodies against class-I-MHC, LFA-1, and CD3 did not block. Pretreatment of erythrocytes with neuraminidase, greatly reduced the SW-inhibitable component of rosetting. The SW effect would appear to be due to a direct interaction of SW with a cell surface structure involved in syngeneic rosette formation rather than the known ability of SW to block the processing of N-linked sugar structures. The results are consistent with cell surface lectins and cell surface sugars playing a role in rosette formation.