Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturityonset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position -1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.