In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.