Aim: Characterization and assessment of the hydration reaction of mineral trioxide aggregate (MTA) Plus exposed to different environmental conditions. Methodology: The specific surface area, surface morphology and characterization of un-hydrated MTA Plus (Avalon Biomed Inc. Bradenton, FL, USA) were investigated. The specific surface area was compared with that of ProRoot MTA (Dentsply International, Tulsa Dental Specialties, Johnson City, TN, USA). The reaction rate was determined using calorimetry, and the hydrated cement was assessed for setting time (determined using an indentation technique), and the set material was characterized using X-ray diffraction analysis, scanning electron microscopy and X-ray energy-dispersive analysis. Atomic ratio plots were drawn to establish the relationship of the hydration products. Three different environmental conditions namely dry or immersed in either water or Hank’s balanced salt solution (HBSS) were used. Results: Mineral trioxide aggregate Plus had a higher specific surface area than ProRoot MTA. The hydration reaction was exothermic. The setting time of MTA Plus was retarded when in contact with fluids (P < 0.001). The setting time was longer when MTA Plus was in contact with HBSS than when in contact with water (P < 0.001). Hydration of MTA Plus resulted in the formation of calcium silicate hydrate, calcium hydroxide, ettringite and monosulfate phases. Bismuth was incorporated in the calcium silicate hydrate structure. The hydration of the core material was not affected by contact with the different solutions but the periphery exhibited microcracking, leaching of calcium hydroxide, partial decalcification of calcium silicate hydrate, inhibition of hydration in contact with the physiological solution. Conclusions: The novel MTA Plus was finer than ProRoot MTA but had a similar chemical composition. MTA Plus in direct contact with fluids exhibited partial decalcification of calcium silicate hydrate in contact with the solution, microcracking and leaching of calcium hydroxide. Interaction with a physiological solution resulted in inhibition of hydration.  (International Endodontic Journal, 46, 831–840, 2013).