Adenylate kinase (AK, EC 2.7.4.3) activity and its compartmentation were investigated in cell cultures of Nicotiana tabacum L. cv. 'Havana 425'. An electrophoretic pattern consisting of six bands was detected by isoelectrofocusing-polyacrylamide gel electrophoresis (IEF-PAGE) and native stain procedures for both directions of the AK-catalysed reaction. Controls using the specific inhibitor adenosine(5')pentaphospho(5')adenosine or omitting AMP in the ATP/ AMP-stain were performed in order to ascertain that the staining was not due to phosphatases or other interfering activities. Mitochondria (80-95% intact) were isolated from cell suspension culture of the line CEH-3. IEF-PAGE revealed only one activity band with a pI of 7.5. Latency tests have shown that this activity is located in the intermembrane space. Chloroplasts (75-85% intact) were isolated from fully grown tobacco leaves. Three activity bands were found in IEF-PAGE with pI of about 4.55, 4.50 and 4.45 respectively. Latency tests indicated different locations within the chloroplast. If plastid morphogenesis in calli was suppressed by norflurazone, all three chloroplast bands lacked. The mitochondrial band and, especially, the band at pI 4.60 were enhanced while the band at pI 4.35 was unchanged. This result confirms the established localisation and suggests the pI 4.60-band as a cytosolic activity.