The small-subunit (SSU) ribosomal RNA (rRNA) gene of the Branchiopod crustacean, Daphnia pulex, was isolated from a phage library and subcloned into the plasmid pBluescript. Determination of the complete nucleotide sequence of this gene revealed it to be 2293 nt in length with a G + C content of 53.7%. This is the longest SSU rRNA gene yet reported from a crustacean. The predicted secondary structure of the rRNA is quite typical for eukaryotes except for length expansion in four regions that are known to be highly variable: V2, V4, V7, and V9. Increases in V4 and V7 were most notable. RT-PCR analysis of these two variable regions showed that they are present in the mature rRNA molecule. Potential secondary structures for these regions are proposed based on energetic criteria. Sequence simplicity analysis of V4 and V7 did not reveal the occurrence of clustered simple sequence motifs. This suggests that replication slippage may not be an important mechanism generating the length expansion in these regions as has been proposed for unusually long regions in insect SSU rRNA genes. On the other hand, there was a pronounced bias in nucleotide composition in the variable regions (G + C = 56.6% in V4 and 60% in V7) which is typical of long SSU rRNA genes in insects. Comparison of the lengths of regions V2, V4, V7, and V9 among diverse arthropods revealed that substantial increases in V4 and V7 tend to co-occur. This suggests the possibility of a functional relationship between these variable regions.