Lipase from Candida rugosa has been immobilized in different formulations of calcium alginate beads, prepared by ionotropic gelation, which differ from each other in CaCl₂ concentration and hardening time, to investigate the effects of immobilization conditions on enzyme properties. Morphological studies on all hydrated beads, performed by SEM equipped with a Peltier plate, revealed a different internal compactness. Despite this, all types of beads had an immobilization yield of 100% measured with the Bradford method and about 94% evaluated from the residual activity of the preparation solutions; moreover, all entrapped biocatalysts catalyzed the complete hydrolysis of p-nitrophenyl acetate, even after one month of storage in distilled water at 4 °C. When the internal microstructure of the beads was more compact, the rate of hydrolysis of the most hydrophobic p-nitrophenyl dodecanoate was halved, probably due to mass transfer limiting effects. The immobilized lipase had better resistance to temperature inactivation than the free form: enzyme residual activity at 50 °C after a week were approximately 70% and 20% for the immobilized and free forms respectively. An excellent recyclability in water at 25 °C of entrapped enzyme was also found, having residual activity greater than 80% at the tenth reaction cycle. The best bead formulation was then used for the resolution of (R)-1-phenylethanol in aqueous solution starting from racemic (R,S)-1-phenylethyl acetate. The enantioselectivity found (E = 10) was slightly higher but did not differ significantly from that of free lipase towards the same substrate (E = 4).