Phosphaturic mesenchymal tumors of the mixed connective tissue type (PMTMCT) are extremely rare, histologically distinctive neoplasms, which cause tumor-induced osteomalacia (TIO) in most cases through the elaboration of a phosphaturic hormone, fibroblast growth factor-23 (FGF23). Rarely, identical tumors without known TIO may be observed. We studied a large group of PMTMCT for expression of FGF23, using a novel reverse transcription polymerase chain reaction (RT-PCR) assay for FGF23 in formalin-fixed, paraffin-embedded tissues. Twenty-nine PMTMCT (17 with and 12 without TIO) and 23 non-PMTMCT (16 various mesenchymal tumors, including 5 chondromyxoid fibroma, 8 chondroblastoma, 1 hemangiopericytoma, 1 aneurysmal bone cyst, and 1 high grade sarcoma; 5 carcinomas; and 2 non-neoplastic tissues) were retrieved. Total RNA was extracted from formalin-fixed, paraffin-embedded sections for RT-PCR analysis. FGF23 was amplified using 3 sets of primers that spanned the intron/exon boundaries to amplify the 3 exons of FGF23 gene (140, 125, and 175 bp). The housekeeping gene phosphoglycerokinase (189 bp) was coamplified to check the RNA quality. Sixteen of 17 (94%) PMTMCT with TIO were FGF23-positive. Nine of 12 (75%) PMTMCT without TIO were FGF23-positive. Two chondromyxoid fibroma and 1 aneurysmal bone cyst were positive; all other non-PMTMCT were negative. We conclude that RT-PCR for FGF23 is a sensitive and specific means of confirming the diagnosis of PMTMCT both in patients with and without TIO. FGF23 gene expression was present in more than 90% of PMTMCT with known TIO, confirming the role of FGF23 in this syndrome. Rare FGF23-negative PMTMCT with known TIO likely express other phosphaturic hormones (eg, frizzled-related protein 4). Our finding of expression of FGF23 in 75% of histologically identical tumors without known TIO confirms the reproducibility of the diagnosis of PMTMCT, even in the absence of known phosphaturia.