Physiological alterations and regulation of heterocyst and nitrogenase formation have been studied in Het Fix mutant strain of diazotrophic cyanobacterium Anabaena variabilis. Het Fix mutant strain of A. variabilis has been isolated by N-methyl-N'-niti-o-N"-nitrosoguanidine (NTG) mutagenesis and was screened with the penicillin enrichment (500 mug ml(-1)). Growth, heterocyst differentiation, nitrogenase and glutamine synthetase (biosynthetic and transferase), (CO2)-C-14-fixation, nitrate reductase (NR), nitrite reductase (NiR), glucose-6-phosphate dehydrogenase (G6PDH), and isocitrate dehydrogenase (IDH) activities, and NO3- NO2-, and NH4+ uptake and whole cell protein profile in different metabolic conditions were studied in the Het Fix mutant strain taking wild-type A. variabilis as reference. Het Fix mutant strain was incapable of assimilating elemental nitrogen (N-2) due to its inability to form heterocysts and nitrogenase and this was the reason for its inability to grow in BG-11(0) medium (free from combined nitrogen). In contrast, wild-type strain grew reasonably well in the absence of combined nitrogen sources and also showed heterocyst differentiation (8.5%) and nitrogenase activity (10.8 etamol C2H4 formed mug(-1) Chl a h(-1)) in N-2-medium. Wild-type strain also exhibited higher NR, NiR, and GS activities compared to its Het(-) Fix(-) mutant strain, which may presumably be due to acquisition of high uptake of NO3-, NO2-, and NH2+. Wild-type strain in contrast to its Het(-) Fix(-) mutant strain also exhibited high level of G6PDH, IDH, and (CO2)-C-14 fixation activities. Low levels of G6PDH and IDH activities in Het Fix mutant strain further confirmed the lack of heterocyst differentiation and nitrogenase activity in the Het(-) Fix(-) mutant strain. NR, NiR, and GS activities in both the strains were energy-dependent and the energy required is mainly derived from photophosphorylation. Furthermore, it was found that de novo protein synthesis is necessarily required for the activities of NR, NiR, and GS in both wild-type and its Het(-) Fix(-) mutant strain.