The nearly one million Alu repetitive elements in the human genome can be grouped into a number of subfamilies. Comparisons between subfamily consensus sequences suggest that Alu evolution is characterized by the sequential amplification and dispersal of a limited number of Alu founder sequences. The S, Sb and Sb1 subfamilies provide an example of such a related series of Alu subfamilies. We have previously demonstrated that adenovirus type 5 and herpes simplex virus type 1 activate RNA polymerase III transcription of endogenous Alu elements in HeLa cells. Here, we report that expression of Alu sequences belonging to the S, Sb and Sb1 subfamilies was activated following infection with these viruses. The data indicate that transpositionally inactive Alu elements can give rise to high levels of pol III transcripts in the presence of appropriate trans-acting factors and demonstrate that the class III promoters of a significant number and variety of Alu sequences are functional in vivo. Multiple subfamilies of Alu sequences were induced in transformed and non-transformed cell types, suggesting that induction of Alu expression may be part of the normal cellular response to viral infection.