A gene of a monomeric hemoglobin, the Pol n component of MV, of a chironomid species, Polypedilum nubifer, was cloned by screening the larval cDNA library with a nucleotide probe corresponding to the N-terminal sequence of purified MV. A clone, 8N, was 755 bp long and comprised a 60 bp 5′ non-coding region, a 209 bp 3′ non-coding region and a 486 bp coding region for 160 amino acids. A comparison of N-terminal sequence of purified MV with that estimated from the DNA sequence of clone 8N, revealed the existence of a signal peptide consisting of 14 residues. This signal peptide was almost exclusively composed of hydrophobic amino acids, suggesting the peptide functions in preglobin transport across the endoplasmic reticulum. The estimated sequence of mature globin of MV showed only 41% of homology to that of CTT-IV, a chromatographically similar monomeric Hb to MV, of an another chironomid species, Chironomus thummi thummi, in a 146 alignment. However, displacements in hydrophilic ⇆ hydrophobic manner were observed only at 28 positions whereas those in hydrophobic ⇆ hydrophobic or hydrophilic ⇆ hydrophilic manner were observed at 45 positions. Furthermore, a comparison of the haem contact positions between these two Hbs showed a remarkable conservance and displacements only in hydrophilic ⇆ hydrophilic or hydrophobic ⇆ hydrophobic manner, suggesting the crucial role of these positions in Hb functionality.