To study the calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, peptidase (denoted as metalloprotease of G. lemaneiformis (GlMP)), a calmodulin-binding protein of G. lemaneiformis was screened using the yeast two-hybrid system. The full complementary DNA (cDNA) and genomic DNA encoding GlMP was obtained by using rapid amplification of cDNA ends (RACE) and genome walking techniques, respectively. The DNA sequence of GlMP consisted of 1,130 nucleotides, containing an open reading frame (ORF) of 462 nucleotides, with an intron of 71 nucleotides. The ORF of the isolated GlMP was predicted to encode 153 amino acids, including the zinc metalloprotease HEXXH consensus motif characteristic of GlMP. Southern blotting indicated that there were two copies of GlMP in G. lemaneiformis. The yeast two-hybrid results showed that the expressed GlMP could interact with CaM, proving that GlMP was a type of calmodulin-binding protein. Next, the transcription of cam and GlMP of G. lemaneiformis was analyzed by using real-time polymerase chain reaction (PCR) under heat shock of 28 and 32 A degrees C. The expression level displayed a cyclical upward trend. These results indicate that GlMP and CaM may play important roles in high-temperature tolerance of G. lemaneiformis.