α1-Adrenergic receptor (AR) activation in cardiac muscle has several different physiological effects that might be mediated through different α1-AR subtypes. Two α1-AR subtypes have been cloned from the rat, the α(1B) and the α(1D); both are present in adult rat heart. A third subtype, the α(1C), cloned from the cow and human, was reported to be absent in the rat. However, we recently found α(1C) mRNA in adult rat heart by using a partial α(1C) cDNA. Thus, all three cloned α1-AR subtypes are present in the heart, but it is unknown whether each is expressed in cardiac myocytes or in cardiac fibroblasts. In the present study, the full-length rat α(1C)-AR was cloned from cultured neonatal cardiac myocytes. α(1C) mRNA transcripts of 3, 9.5, and 11 kb were present in adult rat heart by Northern blot analysis, α(1B)-, α(1C)-, and α(1D)-subtype mRNAs were each present in isolated adult and neonatal cardiac myocytes by RNase protection assay. In addition, cultured neonatal cardiac myocytes expressed the three α1-AR subtype mRNAs. In contrast, none of the α1-AR mRNAs was detected in cultured neonatal cardiac fibroblasts. In addition, α1-ARs were absent in fibroblasts by [3H]prazosin binding and norepinephrine-stimulated [3H]inositol phosphate production. The absence of α1-ARs in cardiac fibroblasts differs from β-adrenergic and angiotensin II receptors, which are present in both cardiac fibroblasts and cardiac myocytes. Three α1-AR subtypes in cardiac myocytes will need to be considered in future studies of the physiological effects of α1-AR activation in cardiac muscle.