The PRB-1b gene codes for a basic-type pathogenesis-related protein of the PR-1 family of tobacco. PRB-1b mRNA accumulation is induced in response to biotic and abiotic elicitors, such as TMV, ethylene, salicylic acid, alpha-amino butyric acid and darkness. In order to determine the location of elements that control dark-regulated PRB-1b gene expression, we tested promoter, transcribed regions and 3'-downstream regions of the gene for their ability to respond to dark induction in transgenic tobacco plants. An ethylene-inducible promoter region of 863 bp was not able to confer dark induction to a beta-glucuronidase reporter gene, while a construct containing the transcribed region of the gene and 3'-downstream sequences, driven by the cauliflower mosaic virus 35S promoter, was correctly dark-regulated. The results indicate that dark-induction of the PRB-1b gene can be controlled by 3'-downstream elements at the transcriptional level or by transcribed sequences at the post-transcriptional level. A circadian clock regulation of the PRB-1b gene was excluded, as fluctuations of PRB-1b transcript levels were not observed in plants placed in constant light or darkness. Subcellular localization of the PRB-1b protein was also determined, in tobacco protoplasts preparations and in cell cultures. The PRB-1b polypeptide was predominantly detected in protoplast vacuoles and was not secreted to the media in cell cultures. These results support an intracellular localization for the PRB-1b protein, as reported for other basic-type components of the pathogenesis-related proteins family.