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Multi-alveolar mammary structures (mammary lobules) were prepared from mammary glands of pseudopregnant rabbits by controlled digestion with collagenase and hyaluronidase. The overall rate of fatty acid synthesis and the proportion of milk-specific fatty acids (C_8:0 and C_10:0) synthesized by these lobules when cultured with insulin, corticosterone and prolactin were measured. Maximum response to physiological concentrations of prolactin (1·1 or 2·2 nmol/l) occurred in the presence of insulin (1·7 µmol/l) and corticosterone (0·58 µmol/l). In general, the results obtained on the effect of progesterone were negative. Though explants showed a ninefold greater response to prolactin per mg DNA than did mammary lobules, the latter have the advantage of being easily prepared for culture in large numbers. Reduction to below 500 µm diameter and culture in conditions which allow cell outgrowth onto plastic limited their response to prolactin. The probable roles of membrane damage by digesting enzymes and of tissue architecture in limiting prolactin response are discussed.