When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.