IL-4 secreting and nonsecreting cells from Th2 cultures have a similar probability of producing IL-4 upon subsequent stimulation, implying that there is stochastic element in IL-4 production by stimulated Th2 cells. Purified IL-4 producers and nonproducers have similar Gata3 and c-maf mRNA expression. Il4 gene accessibility, analyzed by restriction enzyme accessibility (REA) at sites in the promoter, in the second intron (DNase I hypersensitivity sites HSII and HSIII) and in CNS-1 in the two populations was also similar. However, upon TCR stimulation, site V_A, which is 5 kB 3′ of exon 4, displayed a striking increase in accessibility but REA was 2- to 3-fold greater in producers than nonproducers. Cyclosporin A treatment inhibited V_A opening, implying the involvement of NFAT in increased V_A accessibility. Induction of V_A accessibility is sensitive to cycloheximide, suggesting an additional factor(s) is needed. Thus, opening of V_A is a probabilistic event determining which Th2 cells transcribe Il4.