A putative thermostable pectinmethylesterase (TSPME) protein of 36 kDa was isolated from heat-treated citrus finisher pulp. After purification and partial sequencing of the protein, a reverse genetic approach was used to obtain the complete genomic sequence of a new pectinmethylesterase (PME) gene, CsPME4, from Citrus sinensis (L.) Osb. cv. Valencia. The CsPME4 gene contained two exons of 1203 and 690 bp interrupted by a single positionally conserved intron of 1230 bp. A full-length CsPME4 cDNA clone amplified from Valencia orange juice vesicles shared 98% identity with the genomic clone. The encoded protein of the full-length CsPME4 cDNA shared 66 and 39% amino acid identity with the full-length encoded proteins of the citrus PME, CsPME1, and CsPME3, respectively, whereas the predicted mature protein of CsPME4 shared 80 and 61% identity with the predicted mature proteins of CsPME1 and CsPME3, respectively. Southern analysis demonstrated that CsPME4 was present in at least two copies in the Valencia orange genome. Northern analysis revealed that CsPME4 mRNA was accumulated mainly in young and developing tissues of Valencia orange. Several approaches to express recombinant CsPME4 in different systems failed to obtain active protein. Further research will be necessary to successfully express the putative TSPME gene CsPME4 for biochemical characterization.