Increased expression of thyroid hormone responsive protein (THRSP) has been related to higher intramuscular fat (IMF) content in previous studies in cattle. Higher expression of the THRSP gene (1.7-fold, P < 0.05) could be confirmed in M. longissimus of bulls of a Charolais × Holstein F₂-cross with high IMF (6.8 ± 2.4%, n = 10) compared with those with low IMF (1.9 ± 0.5%, n = 10). The presented study aimed at elucidation of the background of the observed expression differences. First, we screened the THRSP locus including 700 bp located 5′ of the transcription start and 1,000 bp containing the 3′-untranslated region (UTR) for single nucleotide polymorphisms (SNPs) with potential effects on gene expression. Only 3 out of 23 previously annotated SNPs were polymorphic in our sample. One new SNP (c.*695T>C) was found in the 3′UTR. None of the SNPs was related to expression of THRSP. To account for the possibility that an elevated amount of THRSP mRNA is an effect of more adipocytes in muscle samples with high IMF, we analyzed adipocytes and myocytes separately. Cells of the respective types were isolated by laser capture microdissection from unstained sections of M. longissimus. Total RNA was extracted from both cell types and cDNA was reversely transcribed from mRNA. Besides THRSP, fatty acid binding proteins (FABP) 3 and 4 were determined as specific markers for muscle and fat cells, respectively. In 2 independent experiments, we found exclusive expression of FABP3 in myocytes and of FABP4 in adipocytes indicating purity of the cell type preparations. THRSP expression was observed in adipocytes but not in myocytes. Moreover, expression of THRSP was not different in subcutaneous fat between both groups of bulls (1.0-fold, P = 0.93) despite a high level of expression. Our results suggest that differences of THRSP mRNA abundance in muscle of cattle with differing IMF result from variable numbers of THRSP-expressing adipocytes in the samples rather than from different activity of the gene.