The sequences required for transcription of Neurospora crassa 5S rRNA genes have been defined using a comprehensive set of deletion and substitution mutations introduced into two cloned genes. An upstream TATA box (consensus TCATAGA) located at -29 to -24, and three internal regions (D, A and C) localized to +19 to +30, +44 to +57 and +73 to +103, respectively, are absolutely required for transcription in vitro. The TATA box fixes the start point of transcription. The A and C regions correspond to the Xenopus 5S gene internal control region but the D region has no Xenopus homologue. The spacing of the three internal elements is very strict but the position of the TATA box can be varied by up to 16 base-pairs.