We sequenced the small-subunit (SSU) rDNA coding region and 1506 group I intron (that interrupts this gene) in Closterium ehrenbergii Menegh. ex Ralfs, Closterium littorale Gay, Cylindrocystis brebissonii (Menegh.) de Bary, Penium margaritaceum (Ehr.) de Bréb. ex Ralfs, and Staurastrum punctulatum de Bréb. and the 1506 intron in Sirogonium sticticum (J.E. Smith) Kütz. (Zygnematales). Reverse transcriptase (RT)/PCR analyses demonstrated that the 1506 introns are not present in vivo in mature rRNA. Splicing analyses in vitro showed, however, that these introns do not catalyze their own excision from the rRNA coding region. Mutation of the conserved G (ωG) to an A at the 3′ terminus of the 1506 intron of Gonatozygon aculeatum Hastings (Zygnematales) restored self-splicing activity. This mutant intron utilized a new 3′ splice site to facilitate autoexcision. We speculate that the 3′ terminus of the wild-type 1506 intron may be a target for a “helper” factor that facilitates the second step of splicing (exon ligation) in vivo in the Zygnematales. Phylogenetic analyses showed congruence of rDNA and intron trees confirming an ancient origin of the 1506 intron in the common ancestor of the Zygnematales. The rDNA trees were compared to those inferred from rbcL sequence analyses. These trees were in general agreement and showed polyphyly of the Mesotaeniaceae and the Zygnemataceae. The 1506 introns contain significant evolutionary signal and provided strong phylogenetic support for groups within the Zygnematales when analyzed alone or in combination with the SSU rDNA coding regions. The secondary structure of 1506 group I introns was studied to identify RNA elements that may be useful as systematic markers. This analysis showed an optional helix found in RNA domain P2 that was lost in a monophyletic group of Desmidiaceae. This helix is found in all other 1506 group I introns, including those interrupting red algal and fungal SSU rDNAs.