Abstract.  The black‐legged tick Ixodes scapularis Linnaeus (Acari: Ixodidae) is an important vector of microbial pathogens. Knowledge of the tick's innate immune response, particularly defensin and other antimicrobial peptides, is important for understanding how microbes survive in this tick. A defensin gene ( sln A) from I. scapularis was obtained by reverse transcription–polymerase chain reaction (RT–PCR) using mRNA extracted from tissues of female ticks. RT–PCR indicated the gene was expressed in the midgut, haemocytes, and fat‐body, although no evidence of a peptide was found. Sequencing a cloned cDNA fragment revealed a 225 bp open reading frame encoding a 74 amino acid pre‐prodefensin, including the putative 38 amino acid mature peptide. Similarity between the defensin amino acid sequences of I. scapularis and Dermacentor variabilis (Say) (Acari: Ixodidae) was 62.2% for the pre‐prodefensin region; for the mature defensins from these two species the similarity was 78.9%, with the six cysteine residues being located in the same relative position. PCR amplification and sequencing of chromosomal DNA suggests that sln A, along with vsn A, the defensin gene from D. variabilis , does not contain any introns. This is in contrast to the defensins described for the soft tick, Ornithodoros moubata ( sensu Walton) (Acari: Argasidae). The role of defensin in the innate immune response of I. scapularis following microbial invasions is discussed.