We have characterized a full-length cDNA (hb-Cg1F) that represents symbiotic mRNA hemoglobin (hb) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb-Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [35S]-labeled probes was compared. The expression of hb-Cg1F gene is induced in young infected host cells prior to the detection of Frankia nifH mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O2 concentration in the cytoplasm of the host cells before the nif genes are expressed.