Antisense oligonucleotides (oligos) have been employed against prostate cancer models in both in vivo and in vitro systems. Most oligos employed by investigators include only a single mRNA-binding site, and target only a single gene. However, some target multiple genes which share sequence homology. Recently, our lab has developed bispecific oligos, which target two different genes not related by sequence homology, and which are able to regulate activity in either the same or in different biological pathways. To date, the effectiveness of bispecific oligos has been evaluated solely by in vitro cell growth inhibition studies. This study evaluates the suppression of targeted mRNA by both mono- and bispecific oligos directed towards the apoptosis regulatory protein BCL-2. The bispecifics used contain binding sites for both the epidermal growth factor receptor (EGFR) and BCL-2 mRNA and differ only in the 5' to 3' tandem orientation. LNCaP cells incubated for 24 hours in the presence of 6.25 muM of oligos suppress the expression of BCL-2 mRNA and support the finding that there is comparable biologic activity produced by both mono- and bispecific oligos in in vitro cell inhibition experiments. For each type of oligo (mono- or bispecific) evaluated, the greatest amount of BCL-2 mRNA suppression approached 100% as produced by the monospecific MR(4) (directed only against BCL-2) and for the bispecifics MR2(4) and MR(42), 86% and 100%, respectively. Suppression was found in duplicate PCR runs employing BCL-2 primers, as well as in multiple agarose gel quantifications. Based upon both inhibition of in vitro growth and bCL-2 expression measured by PCR, we conclude that bispecific antisense oligos directed against both EGFR and BCL-2 mRNAs are at least as effective as a monospecific oligo directed solely towards BCL-2. Therefore the addition of a second mRNA-binding site to these oligos does not prevent activity at the initial site specific for BCL-2.