I-3-Mediated resistance of tomato against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici depends on Six1, a protein that is secreted by the fungus during colonization of the xylem. Among natural isolates of F. oxysporum f. sp. lycopersici are several that are virulent on a tomato line carrying only the I-3 resistance gene. However, evasion of I-3-mediated resistance by these isolates is not correlated with mutation of the SIX1 gene. Moreover, the SIX1 gene of an I-3-virulent isolate was shown to be fully functional in that i) the gene product is secreted in xylem sap, ii) deletion leads to a further increase in virulence on the I-3 line as well as reduced virulence on susceptible lines, and iii) the gene confers full avirulence on the I-3 line when transferred to another genetic background. Remarkably, all I-3-virulent isolates were of race 1, suggesting a link between the presence of AVR1 and evasion of I-3-mediated resistance.