Anabaena sp. PCC 7120 was mutagenized with transposon Tn5-1087b and screened for mutants unable to grow on dinitrogen and altered in heterocyst pattern. Mutant 1801 forms no visible heterocyst within 24h after nitrogen stepdown, and a small number of mature heterocysts within 48h, which amount to 2.8% of total cell number. The mutant is significantly different from the wild type in rate and frequency of heterocyst differentiation. The flanking genomic DNA and the transposon were retrieved from the mutant by restriction of its total DNA with ClaI, recircularization and electroporation into E. coli. As shown with sequencing, the transposon was inserted between Nt. 148 and 149 of alr0099, a gene of unknown function. To prove that the phenotype of the mutant was indeed due to the insertion within alr0099 but not a secondary mutation, we inserted C. K2 cassette, a neomycin resistance marker, into the cyanobacterial genome at the EcoRV site of alr0099 via double homologous recombinations. The reconstructed mutant DR214 showed the same phenotype as 1801. These results indicate that alr0099 or its downstream genes stand for a novel region involved in heterocyst development and pattern formation in the genome of Anabaena sp. PCC 7120.