The purpose of the study was to investigate if the number of goblet cells expanded ex vivo from a conjunctival explant is affected by the biopsy harvesting site on the conjunctiva and the distance from the explant. Conjunctival explants from six regions: superior and inferior bulbus, fornix, and tarsus of male Sprague-Dawley rats were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for eight days. Histochemical and immunofluorescent staining of goblet (CK-7/UEA-1/MUC5AC), stratified squamous, non-goblet (CK-4), proliferating (PCNA) and progenitor (ABCG2) cells were analyzed by epifluorescence and laser confocal microscopy. Outgrowth was measured with NIH ImageJ. For statistical analysis the Mann-Whitney test and Spearman's rank-order correlation test were used. Cultures from superior and inferior fornix contained the most goblet cells as indicated by the presence of CK-7+, UEA-1+ and MUC5AC+ cells. Superior and inferior forniceal cultures displayed 60.8% ± 9.2% and 64.7% ± 6.7% CK-7+ cells, respectively, compared to the superior tarsal (26.6% ± 8.4%; P < 0.05), superior bulbar (31.0% ± 4.0%; P < 0.05), inferior bulbar (38.5% ± 9.3%; P < 0.05) and inferior tarsal cultures (27.7% ± 8.3%; P < 0.05). While 28.4% ± 6.3% of CK-7+ goblet cells co-labeled with PCNA, only 7.4% ± 1.6% of UEA-1+ goblet cells did (P < 0.01). CK-7+ goblet cells were located at a lower concentration close to the explant (39.8% ± 3.1%) compared to near the leading edge (58.2% ± 4.5%; P < 0.05). Both markers for goblet cell secretory product (UEA-1 and MUC5AC), however, displayed the opposite pattern with a higher percentage of positive cells close to the explant than near the leading edge (P < 0.05). The percentage of CK-4+ cells was higher near the explant compared to near the leading edge (P < 0.01). The percentage of CK-7+ goblet cells in the cultures did not correlate with the outgrowth size (r(s) = -0.086; P = 0.435). The percentage of UEA-1+ goblet cells correlated negatively with outgrowth size (r(s) = -0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (r(s) = 0.473; P < 0.05). We conclude that forniceal explants yield the highest number of goblet cells ex vivo and thereby seem to be optimal for goblet cell transplantation. We also suggest that CK-7+/UEA-1- cells represent highly proliferative immature goblet cells. These cells could be important during conjunctival migration as they are mostly located close to the leading edge and their density does not decrease with increasing outgrowth size.