The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.