The 33.5 kDa precursor of the 32 kDa herbicide binding protein is encoded in the chloroplast by the psbA gene. Previous work on the gene and protein did not pinpoint the initiation site of translation in vivo. To resolve this issue, constructs containing full length, truncated or mutated psbA from Solanum nigrum were used as templates for in vitro transcription in an SP6-promoter directed system. The resulting RNAs were then translated in a reticulocyte lysate system. The SDS-PAGE and partial proteolysis patterns of the translation products demonstrate that constructs containing the first ATG codon (Met1) of the psbA open reading frame code for a full length precursor protein (33.5 kDa), irrespective of whether the second ATG codon (Met37) is replaced by site directed mutagenesis or not. However, a 5' deleted gene, lacking the codon for Met1 but containing the codon for Met37, codes for a truncated 29 kDa protein. It is concluded that the 33.5 kDa precursor protein and its 32 kDa maturation product initiate at Met1 of the psbA open reading frame.