Murine insulin-like growth factor 2 antisense (Igf2as) transcripts originate from the opposite strand of the same Igf2 locus as the Igf2 sense mRNA. The Igf2, insulin 2 (Ins2), and H19 genes form a cluster of imprinted genes on chromosome 7. Loss of imprinting of IGF2 in humans is associated with Beckwith-Wiedemann syndrome and Silver-Russell syndrome, as well as with Wilm's tumor and colorectal cancer. We developed a RNA-FISH protocol to detect Igf2as and Igf2 transcripts. The results from the RNA-FISH were confirmed with quantitative real-time PCR and clearly indicate that the Igf2as transcripts are predominantly located in the cytoplasm of C2C12 cells. In a polysome association study, we showed that the Igf2as sedimented with polysomes in a sucrose gradient. The cellular localization of Igf2as transcripts together with polysome fractionation analysis provides compelling evidence that the Igf2as is protein coding.