Protopine biosynthesis in Fumaria rostellata and Fumaria officinalis cell suspensions was investigated. For the first time, we reported for calli and cell suspensions obtained from F. rostellata and F. officinalis. Callus induction was initiated on a Murashige and Skoog medium, supplemented with sucrose and various concentrations of plant growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The best morphological characteristics, growth behavior, and protopine biosynthesis were observed for two callus lines (5FRL14 and 12FOL1) cultivated under submerged conditions, at low concentration of 2,4-D (0.2 and 0.5 mg/L) and higher concentration of BAP (2.0 and 3.0 mg/L). The maximal yield of protopine was accumulated from cell suspension of F. rostellata (line 5FRL14) cultivated under illumination-49.6 mg/L. Time courses of utilization of sucrose, ammonium, nitrate, and phosphate ions in cultural liquid and acetylcholinesterase inhibitory activity of alkaloid extracts of studied suspensions are also presented.