To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C- raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C- raf gene expression in vitro , and antitumor activity in vivo . Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C- raf RNA sequences and reduced inhibitory activity against C- raf expression in vitro and tumor growth in vivo . Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.