The organization of the intergenic spacer of a 9.04 kb tomato ribosomal RNA gene (rDNA) was determined. The 3258 bp spacer contains two major repeat elements enclosing a region which includes 351 bp of an 81.8% A --T rich sequence. A block of nine 53 bp repeats begins 388 bp downstream from the 3' end of the 25S rRNA. The A--T rich domain is followed by a block of six 141 bp repeats terminating 818 bp upstream from the 5' end of the 18S rRNA. Major pre-rRNAs of 7.6 and 6.5 kb were observed by Northern hybridization analysis. The 5' termini of these RNAs were identified through combined S1 nuclease and primer extension analyses. The 7.6 kb RNA is likely to be the primary transcript; its 5' terminus lies within a sequence motif. TATA(R)TA(N)GGG, conserved at the termini of transcripts mapped in three other plant species. The 6.5 kb RNA is interpreted as a 5' end processed transcript derived from the 7.6 kb RNA. Comparative analysis of transcribed sequences revealed a 25 bp domain of the intergenic spacer which is relatively conserved among five plant species. The conservation of spacer sequences in plants is in contrast to the extensive sequence divergence of the intergenic spacer in other non-plant systems and suggests a conserved function directed by these sequences.