The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, and Cucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5' region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes. The two investigated Cucurbitaceae species show a G + C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G + C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G + C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G + C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.