Leghemoglobin is a major component of the nitrogen-fixing nodules formed by legumes in association with bacterial symbionts of the genusRhizobium. It is thought to be involved in regulating the oxygen tension within nodules. In a series of Southern blot experiments using cloned soybean leghemoglobin cDNAs as hybridization probes, cross-hybridizing sequences have been detected in legumes closely related to soybean (members of the Leguminosae subfamily Papilionoideae), as well as in a distantly related legume not reported to be nodulated (subfamily Caesalpinioideae). With the same probes, the presence of cross-hybridizing sequences has also been detected in plants outside the Leguminosae, including two nitrogen-fixing non-legumes and one species which is not nodulated. These results suggest that the genes for oxygen-binding proteins may be more widely dispersed than previously thought.