A systematic study of the influence of carrier particle size (500-850 μm) and enzyme load (26,200-66,100 lipase activity units (LU)/g dry carrier) on the content and activity of Candida antarctica lipase B (CALB) immobilized by adsorption onto macroporous poly(methyl methacrylate) (PMM) and polystyrene (PS) carriers was conducted. Furthermore, localization of CALB on the carrier was investigated by light and fluorescence microscopy of freeze microtome sliced catalyst particles. Fluorescence microscopy showed localization of enzyme in an outer rim of 50-85 and 10-20 μm thickness for the PMM and PS catalysts, respectively, whereas no rim was observed in the absence of enzyme. Statistical analyses showed that carrier type was the major effect in determining the activities of the catalysts, with enzyme load being the second most significant effect and particle size also exerting a significant, yet smaller, effect. The PMM catalysts showed higher activities compared to PS catalysts, possibly indicating that the microenvironment interactions of CALB with the PMM are more favorable than with the PS carrier, resulting in a higher specific enzyme activity. Furthermore, smaller particles and higher enzyme load had a positive influence on the activities within the investigated ranges, and the carrier type and enzyme load interaction was statistically significant (p < 0.001).