We have extended our previous studies of fra(X) expression in somatic cell hybrids to the analysis of a carrier female with a low level of expression and to an unaffected, transmitting male who shows no expression in lymphocytes or lymphoblasts. Optimum conditions for fra(X) expression was treatment with 10(-8) M FUdR for 16 hours. In recent experiments, addition of 2.2 mM caffeine 2 hours before harvest was found to increase expression consistently. Two clones from the carrier female containing the fra(X) chromosome but not the normal X showed expression of 2-4%, indicating that expression in heterozygous females is not influenced by the presence or absence of the normal X. Expression rate was increased to 20% by exposure to FUdR plus caffeine. Analysis of hybrids containing only the fra(X) in an inactive state, and after reactivation by 5-azacytidine, showed no change in the frequency of expression. A hybrid clone from the nonexpressing, transmitting male containing only the X and chromosome 13, showed expression ranging from 2% without caffeine to 12% with caffeine in three different experiments. The ability to induce fra(X) expression in hybrid from this nonexpressing male may be explained in one of several ways: 1) a second mutation has occurred, 2) an autosomal suppressor locus was lost, or 3) the hamster genome or unusually short cell cycle lowers the threshold for expression, particularly in the presence of caffeine.