Two calmodulins are synthesized during differentiation of Naegleria gruberi from amoebae to flagellates; one remains in the cell body and the other becomes localized in the flagella. The single, intronless, expressed gene for flagellar calmodulin has been cloned and sequenced. The encoded protein is a typical calmodulin with four putative calcium-binding domains, but it has an amino-terminal extension of 10 divergent amino acids preceding conserved calmodulin residue 4. The transcripts encoding flagellar calmodulin and flagellate cell body calmodulin are clearly divergent. Expression of the flagellar calmodulin gene is differentiation-specific; its mRNA appears and then disappears concurrently with those encoding flagellar alpha- and beta-tubulin. Three provocative sequence elements are shared among these unrelated coexpressed genes: (i) a palindromic DNA sequence element is found in duplicate or triplicate upstream to each transcribed region; (ii) a perfect 12-nucleotide match is found near the AUG start codon of flagellar calmodulin and alpha-tubulin; and (iii) the novel amino-terminal extension of flagellar calmodulin contains a 5-amino-acid element similar to the amino terminus of flagellar alpha-tubulin. These shared sequence elements are proposed to have roles in differentiation, possibly in regulation of transcription, mRNA stability, and localization of these proteins to flagella.