We have studied the expression of phospholamban during the early development of chick embryos by in situ hybridization and have compared it to that of α-cardiac and α-skeletal actin. In adult cross-striated muscles there is only one phospholamban gene and it is expressed exclusively in the heart and slow muscles. In the heart phospholamban transcripts were first detected at stage 14 in the region of presumptive ventricle and at stage 20 in the atrium. In the myotomal portion of the somites phospholamban mRNA was first detected at stage 20, which lagged behind the appearance of the α-actins. In the limb rudiments all three mRNAs were barely detectable through stage 24, but increased by stage 28 +. However, quantitative analysis of signal intensity at stage 28 + indicated that less phospholamban mRNA is present in the limb bud than in the myotome since for phospholamban the ratio of the signal density in the myotome to that in the limb rudiments was about twice the value of the ratio determined for the α-actins. Northern blot analysis of embryonic day 11 chick fast pectoralis muscle showed that phospholamban mRNA was not detected in vivo while α-cardiac actin mRNA was. Moreover, no phospholamban mRNA was detected in primary cultures derived from pectoralis muscle of the same age. In concert with previous observations that phospholamban is not detectable at stage 30–32 in wing or thigh muscle, these results suggest that phospholamban mRNA is expressed independently of the α-actins in the limb buds during early myogenesis. © 1993 wiley-Liss, Inc.