Abstract This paper reports the expression and localization of linamarase in roots of two cassava (Manihot esculentaCrantz) cultivars of low and high cyanide. Two different patterns of linamarase activity were observed. In the low-cyanide type, young leaves displayed very high enzyme activity during the early plant growing stage (3 months), whereas in root peel, the activity increased progressively to reach a peak in 11-month-old plants. Conversely, in the high-cyanide cultivar (HCV), root peel linamarase activity decreased during the growth cycle, whereas in expanded leaves linamarase activity peaked in 11-month-old plants. The accumulation of linamarin showed a similar pattern in both cultivars, although a higher concentration was always found in the HCV. Linamarase was found mainly in laticifer cells of petioles and roots of both cultivars with no significant differences between them. At the subcellular level, there were sharp differences because linamarase was found mainly in the cell walls of the HCV, whereas in the low-cyanide cultivar, the enzyme was present in vacuoles and cell wall of laticifer cells. Reverse transcriptase-PCR on cassava tissues showed no expression of linamarase in cassava roots, thus, the transport of linamarase from shoots to roots through laticifers is proposed.