Cotyledon protoplasts were isolated from 16-18-day-old in vitro grown seedlings of 9 Brazilian and 3 Hungarian pepper varieties and hybrids. Large numbers (average 9.59 X 106 protoplasts g 14 fresh weight) of highly viable (average 87.0%) protoplasts were released using a pectocellulolytic enzyme mixture. Protoplasts were cultured in K8p mediuni u...sing an alginate disc embedding method. The osmotic pressure of the medium surrounding the alginate-embedded protoplasts was reduced by replenishing the liquid medium at K8p:K8 ratios of 1:0. 2:1, 1:1 in the first. second, and third week, respectively. Initial plating efficiency (IPE) average was 38.5% and after 21 days protoplasts reached microcolonies (15-20 cells) stages. Microcolonies were transferred after 3-4 weeks to a MS-based medium supplemented with 1.0 mg I-1 zeatin, 3.0% (w/v) sucrose, 0.24% (w/v) phytagel and pH 5.8, whereupon they formed callus. Final plating efficiency (FPE) average was 0.29% at a plating density of 1.0 x 105 protoplasts Protoplast-derived calli were cultured on a range of MS-based media supplemented with either BAP, IAA, TDZ; and zeatin. No morphogenic response was observed in any genotype investigated.