The effects of two proline analogues,l‐3,4‐dehydroproline andl‐azetidine‐2‐carboxylic acid, on collagen synthesis by cultured 3T6 fibroblasts have been studied. Prolyl hydroxylase activity was partially inhibited in cells cultured with dehydroproline for 24 h, resulting in the synthesis of collagen in which the proline was underhydroxylated. Azetidine had no effect on prolyl hydroxylase and less effect on the degree of hydroxylation of proline. Fibroblasts grown in the presence of either analogue and fixed in‐situ contained greatly distended cisternae of the rough endoplasmic reticulum. Proline analogues otherwise caused few ultrastructural changes in the cells. Treated cells which had been handled more roughly during preparation for electron microscopy contained many large cytoplasmic vacuoles in addition to dilated cisternae. Our results indicate that the major effect of the proline analogues was the inhibition of prolyl hydroxylation. However, electron microscopy of the treated cells revealed hitherto unreported cytopla