Fibroblast growth factor-2 (FGF-2) or basic FGF is a multifunctional protein that, through interaction with specific cell surface receptors, plays important roles in the growth and development of tissues and organs. Thus, considerable attention has focused on the control of FGF-2 gene expression, including assessments of RNA levels through blotting and the use of radiolabeled FGF-2 cDNA probes. Multiple transcripts of different sizes have been reported for FGF-2 by this approach, however, more recent evidence indicates that at least one of these RNAs of about 1.5 kb, is not an authentic FGF-2 transcript. A major band of 4.7 kb and a minor band of 6.1 kb were detected in total rat glial tumor cell RNA, using the ‘intact’ rat ovarian FGF-2 cDNA as a probe at high stringency. This cDNA contains both coding and 5′-untranslated sequences. Although the 6.1 kb transcript levels were increased in RNA enriched for polyadenylated species, the levels of the 4.7 kb band were decreased and also shared a mobility with 28S RNA. A truncated FGF-2 cDNA probe, containing coding but not 5′-untranslated sequences, detected the 6.1 kb transcript but failed to see the 4.7 kb band. The domain responsible for detecting the 4.7 kb band was localized to a G/C-rich region containing 5′-untranslated sequences, by using different fragments of the rat FGF-2 gene, including coding and upstream flanking DNA, as probes. The degree of similarity between sequences of this G/C-rich region of the FGF-2 gene and 28S RNA from rat, human and mouse was sufficient to predict strong cross hybridization. This was confirmed by the detection of a 4.7 kb band in mouse heart RNA with the ‘intact’ but not truncated rat FGF-2 cDNA probes; a 6.1 kb mouse FGF-2 transcript was detected with both probes. These data indicate that the 4.7 kb RNA detected is not a bona fide FGF-2 transcript, and most likely represents cross hybridization with abundant 28S RNA through G/C-rich non-coding sequences present in the ‘intact’ rat FGF-2 cDNA. However, sequence comparisons suggest that this result may be the case for other species and might not be restricted to the rat FGF-2 cDNA.