Karyotyping in combination with fluorescence in situ hybridization (FISH) on tomato pachytene chromosomes allowed identification and mapping of a major 45S (5.8S, 18S and 25S) rDNA site on the satellite of 2S and four minor loci, each at a proximal knob on 2L, 6S, 9S and 11S. Thus, the 45S rDNA loci are all located in heterochromatic regions. The five 45S sites are all transcriptionally active as evidenced by a maximum of ten nucleoli in meiotic cells at telophase or interphase. The 45S rDNA loci, as well as the 5S rDNA locus on 1S, were highlighted by chromomycin A3, a GC-specific DNA ligand; this result is consistent with the high GC content of the rDNA genes. Satellite size varied dramatically between genotypes. Enzymatic maceration of tomato anthers followed by squashing in acetocarmine produced high quality chromosomal preparations and subsequent FISH images by reducing the strong autofluorescence inherent in the nucleolus and cytoplasm of tomato meiotic cells. Our protocol has potential in the construction of an integrated cytological, classical and molecular map of tomato.