We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that α-cardiac actin mRNA levels increased during cell differentiation while β-actin mRNA levels declined. Nonmuscle cells displayed β-actin mRNA signal localized at the cell periphery, while α-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in α-cardiac actin mRNA expression after 72 h of infection. In contrast, β-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ β-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.