The gene encoding fibroin light chain protein (FibL) is specifically expressed in the posterior silk gland of silkworm and repressed in other tissues. The binding sites of several transcription factors involved in the silk gland transcription specificity of fibl promoter have been recognized, including SGFB, PSGF and BMFA. Here we report the leak expression of the enhanced green fluorescent protein (EGFP) reporter gene in tissues other than the posterior silk gland in vivo when under the control of a shortened fibl promoter with deletion of the 5′ terminal 41 bp sequence, which is located at −650 nt to −610 nt upstream of the fibl transcription starting site. Assay of silk gland specificity of the promoters was performed by observation of green fluorescence in tissues of silkworm larvae following inter-haemocoelic injection of recombinant Autographa californica multiple nuclear polyhedrosis virus carrying the EGFP reporter gene controlled by different lengths of fibl promoters. Our results indicated that availability of the binding sites of several known factors, including SGFB, PSGF and BMFA, is not sufficient for intact silk gland transcription specificity of fibl promoter, and there are possible inhibitor binding sites in the 41 bp sequence (−650 nt to −610 nt) upstream of the transcription starting site which may be required to repress the activity of fibl promoter in other tissues.