Inhibition of expression of specific genes by means of antisense RNA is widely used, although little information is available regarding conditions that affect the efficacy of inhibition. In this study, inhibition of granule-bound starch synthase (GBSS), a key enzyme in starch biosynthesis, is used as a model system. Eleven antisense constructs derived from the full-length GBSS cDNA, the genomic GBSS coding region (gDNA) or fragments of each of these sequences, were analysed with respect to their inhibitory effect. Introduction of full-length gDNA constructs yielded a lower percentage of transgenic clones showing complete inhibition than did introduction of the full-length cDNA constructs. This may be caused by a lower antisense binding capacity of the former due to the relatively low GC content in intron sequences present in the gDNA constructs. The presence of multiple T-DNA insertions was related to a higher degree of inhibition. Putative polyadenylation signals on the antisense strand of the GBSS gene resulted in a premature stop of transcription of some of the antisense genes, as demonstrated by the expression of smaller antisense RNA transcripts. Introduction of antisense constructs driven by the promoter of the (target) GBSS gene resulted in a higher percentage of clones with complete inhibition than introduction of antisense constructs driven by the 35S CaMV promoter. Complete antisense inhibition was achieved in 25% of the clones carrying the antisense construct pKGBA50, which is based on the GBSS promoter and the full-length GBSS cDNA. Thus, it is concluded that the use of pKGBA50 is very suitable for the modification of the composition of potato tuber starch via antisense RNA.