Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence. Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters. The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp. One spacer of 9 bp length was varied at a single position. IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects. In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.